Sizable tunable enrichment platform for capturing nano particles in a fluid

ABSTRACT

The invention provides enrichment platform devices for size-based capture of particles in solution. The enrichment platform device is useful for label-free capture of any particle. The invention relates to enrichment platform devices using nanowires and vertically aligned carbon nanotubes. The invention provides methods for making the enrichment platform devices. The invention provides methods for using the enrichment platform devices for filtering particles, capturing particles, concentrating particles, and releasing viable particles.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application to co-pending U.S.application Ser. No. 15/213,128 filed on Jul. 18, 2016, which claimspriority to U.S. Provisional Patent Application No. 62/193,876, filedJul. 17, 2015, the contents of each is incorporated by reference hereinin its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under Grant No. CA174508and TR000127 awarded by the National Institutes of Health, under GrantNo. FA9550-12-1-0035 awarded by the United States Air Force and underHatch Act Project No. PEN01607 awarded by the United States Departmentof Agriculture. The Government has certain rights in the invention.

BACKGROUND OF THE INVENTION

The traumatic impact of viral infectious diseases was witnessed in the1918 influenza pandemic, the ongoing HIV/AIDS pandemic, the most recentEbola outbreak (Fauci A. S. et al., New Engl J Med, 2012, 366:454-461),and more. Rapid isolation, identification, and characterization ofviruses from field samples are critical in the prevention of epidemics(Pennington H., Nat Rev Micro, 2004, 2:259-262). Small footprintlab-on-a-chip systems promise to rapidly analyze samples of smallquantity with high sensitivity at points of care (Whitesides G. M.,Nature, 2006, 422:368-373). Although many advanced virus detectionmethods have been reported, there has yet to be a high efficiency samplepreparation system (Ritzi-Lehnert M., Expert Rev Mol Diagn, 2012,12:189-206; Sin M. L. Y. et al., Expert Rev Mol Diagn, 2014,14:225-244). Most previous work employed antibodies or ligands tocapture known nanoscale target like viruses (Stern et al., Nature, 2007,445:519). Existing virus sample preparation systems utilizing, e.g.immune-based capture, which requires foreknowledge of virus strain ormembrane-based filtration, suffers from low efficiency and capacity dueto non-uniformity of pore size and low porosity, as well as a lack ofdownstream virus analysis integration and lowers accessibility to thepublic. Label-free methods will be highly desirable for unknown viruses.In addition, it is difficult to release the captured NPs for furtheranalysis.

There is a need for an improved method of label-free capture for smallparticles like viruses. The present invention meets this need.

SUMMARY OF THE INVENTION

The invention provides enrichment platform devices for size-basedcapture of particles in solution. The enrichment platform device isuseful for label-free capture of any particle. The invention relates toenrichment platform devices using nanowires and vertically alignedcarbon nanotubes. The invention provides methods for making theenrichment platform devices. The invention provides methods for usingthe enrichment platform devices for filtering particles, capturingparticles, concentrating particles, and releasing viable particles.

In one aspect, the invention relates to an enrichment platform devicefor size-based, label-free capture of particles in sample solution, thedevice comprising: a substrate; vertically-aligned carbon nanotubesarrays (VACNT); and a cover having at least one inlet and at least oneoutlet; wherein the VACNT is attached to the substrate, the cover bondsto the substrate to seal the VACNT within the cover, and sample solutionenters via the at least one inlet, passes over the VACNT, and exits viathe at least one outlet, whereupon particles in the sample solution arecaptured by the gaps between the VACNT based on size.

In one embodiment, the substrate comprises material selected from thegroup consisting of: silicon, glass, sapphire, metals, and polymers. Inone embodiment, the cover comprises material selected from the groupconsisting of: plastics, metals, glass, sapphire, polymers, andpolydimethylsiloxane (PDMS). In one embodiment, the cover is removable.

In one embodiment, the VACNT comprise single-walled CNT, double-walledCNT, multi-walled CNT, and combinations thereof. In one embodiment, theVACNT is nitrogen-doped VACNT, boron-doped VACNT, silicon-doped VACNT,aluminum-doped VACNT, phosphorus-doped VACNT, lithium-doped VACNT, andcombinations thereof. In one embodiment, the VACNT are separated by agap size between 1 nm and 500 nm.

In one embodiment, the device is a microfluidic device. In oneembodiment, the device is a handheld device.

In another aspect, the present invention relates to an enrichmentplatform device for size-based, label-free capture of particles insample solution, the device comprising: a substrate comprising aplurality of channels; a cover having at least one inlet and at leastone outlet; and a plurality of nanowires; wherein the plurality ofnanowires are attached to the substrate within the channels, the coverseals the plurality of channels, and sample solution enters via the atleast one inlet, passes through the plurality of channels, and exits viathe at least one outlet, whereupon particles in the sample solution arecaptured by the gaps between the plurality of nanowires based on size.

In one embodiment, the substrate comprises material selected from thegroup consisting of: silicon, glass, sapphire, metals, and polymers. Inone embodiment, the cover comprises material selected from the groupconsisting of: plastics, metals, glass, sapphire, polymers, andpolydimethylsiloxane (PDMS). In one embodiment, the cover is removable.

In one embodiment, the nanowires comprise materials selected from thegroup consisting of: silicon, zinc, zinc oxide, and nickel. In oneembodiment, each nanowire is separated by a gap size between 1 nm and500 nm.

In one embodiment, the device is a microfluidic device. In oneembodiment, the device is a handheld device.

In another aspect, the present invention relates to a method of makingan enrichment platform device comprising VACNT, the method comprising:depositing a metal catalyst thin film on a substrate by e-beamevaporation and lift-off process; patterning the metal catalyst thinfilm using lithography; depositing CNT precursor material on the metalcatalyst thin film using chemical vapor deposition (CVD) to createVACNT; and bonding a cover to the substrate to encase the VACNT.

In one embodiment, the metal catalyst thin film is an iron-catalyst thinfilm, a nickel-catalyst thin film, or a cobalt-catalyst thin film. Inone embodiment, the metal catalyst thin film thickness is adjusted totune gap size, diameter, and density of the VACNT. In one embodiment,increasing the metal catalyst thin film thickness increases the VACNTgap size. In one embodiment, the gap size is tuned between 1 nm and 500nm. In one embodiment, increasing the metal catalyst thin film thicknessincreases the VACNT diameter. In one embodiment, increasing the metalcatalyst thin film thickness decreases the VACNT density.

In one embodiment, the precursor comprises doping material. In oneembodiment, the doping material is selected from the group consistingof: nitrogen, boron, silicon, aluminum, phosphorus, and lithium.

In another aspect, the present invention relates to a method of makingan enrichment platform device comprising porous silicon nanowires(PSNWs), the method comprising: depositing a thin photoresist layer on asilicon substrate; patterning the thin photoresist layer by removingphotoresist using lithography; etching a channel in the substrate wherephotoresist is absent; depositing a thicker layer of photoresist on theexisting layer of photoresist; depositing silver nanoparticles (SNP)within the etched channel; reacting with the SNP as a catalyst toperform silicon etching to form PSNWs; removing the photoresist and SNP;and bonding a cover to the substrate to encase the PSNWs.

In one embodiment, the etching is deep reactive-ion etching (DRIE),metal assisted silicon etching, or wet etching. In one embodiment, thegap size of the PSNWs is tuned by adjusting SNP deposition time. In oneembodiment, increasing SNP deposition time increases PSNW gap size. Inone embodiment, the gap size is tuned between 1 nm and 500 nm.

In another aspect, the present invention relates to a method offiltering particles of a specific size out of a solution, the methodcomprising: fabricating an enrichment platform device comprising a gapsize matching the size of the particle to be filtered; and passing thesolution through the filter.

In another aspect, the present invention relates to a method ofcapturing particles of a specific size out of a solution for analysis,the method comprising: fabricating an enrichment platform devicecomprising a gap size matching the size of the particle to be filtered;passing the solution through the filter; and analyzing the capturedparticles in the enrichment platform device.

In another aspect, the present invention relates to a method ofcapturing viable particles of a specific size out of a solution andreleasing the same viable particles for analysis, the method comprising:fabricating an enrichment platform device comprising a gap size matchingthe size of the particle to be filtered; passing the solution throughthe filter; removing the enrichment platform device cover; releasing thecaptured viable particles from the enrichment platform device; andanalyzing the released viable particles.

In one embodiment, the particles are released by scratching the devicesurface. In one embodiment, the particles are released by degrading thedevice nanostructures. In one embodiment, the solution is derived from apatient. In one embodiment, the captured particles are used to diagnosethe patient as being host to the captured particles. In one embodiment,a patient being diagnosed as hosting the captured particles isindicative of having a disease.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description of preferred embodiments of theinvention will be better understood when read in conjunction with theappended drawings. For the purpose of illustrating the invention, thereare shown in the drawings embodiments which are presently preferred. Itshould be understood, however, that the invention is not limited to theprecise arrangements and instrumentalities of the embodiments shown inthe drawings.

FIG. 1, comprising FIG. 1A and FIG. 1B, depicts an illustration of avertically-aligned carbon nanotube (VACNT) microfluidic device. FIG. 1Adepicts a device wherein the VACNT are bonded to substrate on the bottomand to cover on the top. FIG. 1B depicts a device wherein the VACNT arefreestanding and are bonded to substrate only on the bottom.

FIG. 2A through FIG. 2C depict the design and operation of the pSiNWsforest based device for viral isolation. FIG. 2A: size of variousbiological molecules in the micro and nano scale. FIG. 2B: illustrationsof pSiNWs forest based microfluidic device showing the overallmicrofluidic design in 3D, the location of the pSiNWs forests (topinset, cross-sectional view), and nanoparticles captured inside thepSiNWs forest (bottom inset). FIG. 2C: photo of a prototype device.

FIG. 3A through FIG. 3H depict the synthesis and characterization ofpSiNWs. FIG. 3A: Sketch of synthesizing pSiNWs showing larger SNPsdefining the inter-wire spacing and tiny SNPs generating porousstructures on individual silicon nanowire. Black solid arrows indicatereactant fluoride ions coming to the surface. Red dash lines indicateproduct silicon hexafluoriode anions leaving the surface into the bulk.FIG. 3B: Scanning Electron Microscope (SEM) of SNPs and channel bottom(bar: 500 nm, insert bar: 200 nm). FIG. 3C: SEM images of pSiNWs onchannel bottom (bar: 500 nm, insert bar: 200 nm). FIG. 3D: SEM images ofpSiNWs on side wall (bar: 2 μm). FIG. 3E: SEM images of cross-sectionview of pSiNWs (bar: 2 μm, two inserts' bar: 500 nm). FIG. 3F: Sizes ofSNPs and inter-wire spacing of pSiNWs forests versus the silvernanoparticle deposition time. FIG. 3G, FIG. 3H: Distribution of theinter-wire spacing of pSiNWs with silver nanoparticle deposition time of45 s (FIG. 3G) and 60 s (FIG. 3H), respectively.

FIG. 4 depicts an exemplary fabrication process of the pSiNWmicrofluidic device. 1. Patterning photoresist SPR 3012 on highconductivity silicon wafer by photolithography. 2. Patterningmicrofluidic channels on the silicon wafer by DRIE with the SPR 3012mask. 3. After DRIE, as SPR 3012 mask could not protect the surface ofsilicon as well as AZ p4620 during synthesizing pSiNWs (FIG. 5), SPR3012 was replaced by AZ 4620. Removing photoresist SPR 3012 innanostrip. 4. Protecting outside surface of channels by patterning thickphotoresist AZ p4620 using photolithography. 5. Depositing SNPs on thebottom and side walls of the channels in the solution of 0.085% AgNO3and 9.8% HF. 6. Etching pSiNWs forests with metal-assisted etching inthe solution of 0.35% H₂O₂ and 9.8% HF; 7. Removing photoresist AZ 4620in acetone and SNPs in the silver etchant; 8. Sealing the microfluidicchannels with PDMS after surface treatment of silicon wafer and PDMSwith oxygen plasma.

FIG. 5A and FIG. 5B depict the protective effects of SPR 3012 mask (FIG.5A) and AZ P4620 mask (FIG. 5B) during synthesizing PSNWs (bar: 200 μm).When using SPR 3012 mask after DRIE etching, silicon surface outside thechannels was damaged by the etching solution. While using AZ 4620 mask,silicon surface outside the channels was fine.

FIG. 6A through FIG. 6D depict fluid flow in the cross section (labeledbox) of the 20 μm depth channels. The FEM model, which is a singlechannel with five repeatable units, was simulated in Comsol. FIG. 6A:Schematic of the channel. FIG. 6B: Velocity field along the channel (xdirection, V_(x)). FIG. 6C: Velocity field in the horizontal plane (ydirection, V_(y)). FIG. 6D: Velocity field in the vertical plane (zdirection, V_(z)).

FIG. 7A through FIG. 7D depict fluid flow in the cross section (labeledbox) of the 40 μm depth channels. The FEM model, which is a singlechannel with five repeatable units, was simulated in Comsol. FIG. 7A:Schematic of the channel. FIG. 7B: Velocity field along the channel (xdirection, V_(x)). FIG. 7C: Velocity field in the horizontal plane (ydirection, V_(y)). FIG. 7D: Velocity field in the vertical plane (zdirection, V_(z)).

FIG. 8A through FIG. 8D depict fluid flow in the cross section (labeledbox) of the 60 μm depth channels. The FEM model, which is a singlechannel with five repeatable units, was simulated in Comsol. FIG. 8A:Schematic of the channel. FIG. 8B: Velocity field along the channel (xdirection, V_(x)). FIG. 8C: Velocity field in the horizontal plane (ydirection, V_(y)). FIG. 8D: Velocity field in the vertical plane (zdirection, V_(z)).

FIG. 9 depicts the forces on nanoparticles in a meandering channel.

Wall lift force: F _(L) =f(β)ρ_(f) v _(m) ² a ⁴ /D _(H) ²

Dean force:

${F_{D} = {{1.0}8 \times 10^{- 3}{\pi\mu}\;{De}^{1.63}a}},{{De} = {{Re}\sqrt{\frac{D_{H}}{2R}}}}$

Stokes drag: F_(s)=6πμav

Here, ƒ(β) is the wall lift coefficient, ρ_(ƒ) is the density of thefluid, v_(m) is fluid velocity in z direction, a is the nanoparticleradius, D_(H) is the hydraulic diameter of the channel, μ is the dynamicviscosity of the fluid, v is the velocity in βdirection, De is Deannumber, Re is Reynolds number, R is the curvature radius of the channel.In the device, the velocity of the fluid is 0.011 m/s. The height of thechannel was 20 μm. The width of the channel was 100 μm. The curvatureradius was 100 μm. The nanoparticle radius was about 50 nm, Under theseconditions, the wall lift force was about 10⁻¹⁹ N, and the Dean forcewas about 7.7×10⁻¹⁵ N. So the Dean force was larger than the wall liftforce by 3 orders of magnitude. (Ho, B. P. et al., Journal of FluidMechanics 1974, 65:365-400; Asmolov, E. S., Journal of Fluid Mechanics1999, 381:63-87; Matas, J.-P. et al., Journal of Fluid Mechanics 2004,515:171-195).

FIG. 10A through FIG. 10I depict the capture of nanoscale particlesinside the pSiNWs microfluidic devices with the effect of the Dean flow.FIG. 10A through FIG. 10C: FEM simulations showing velocity field on thecross-sectional planes of meandering flow channels with channel depthsof 20, 40 and 60 μm, respectively. 0 and 100 μm point the outer andinner rim of channel, respectively. FIG. 10D: Capture efficiency of 75nm and 400 nm nanobeads in channels with 20, 40 and 60 μm height,respectively. FIG. 10E: Capture of 75 nm green nanobeads showing topviews of the pSiNWs flow channels before, during, and after injectingnanobeads (bar: 200 μm); FIG. 10F: Capture of 400 nm blue nanobeadsshowing top views of the pSiNWs flow channels before, during and afterinjecting nanobeads (bar: 200 μm); FIG. 10G: SEM image of captured 75 nmnanobeads (bar: 150 nm). FIG. 10H: Capture efficiency of 75 nm and 400nm nanobeads under different flow rates. FIG. 10I: Capture efficiency of75 nm and 400 nm nanobeads versus number of run times of the same samplein the same device.

FIG. 11A through FIG. 11C depict the degradation of pSiNWs and releaseof captured nanobeads. FIG. 11A: SEM images of pSiNWs s from the topview after soaking in PBS for 0, 24, 48 and 72 hours (bar: 1 μm). FIG.11B: SEM images of pSiNWs from the side view after soaking in PBS for 0,24, 48 and 72 hours (bar: 2 μm). FIG. 11C: Releasing nanobeads bydegrading pSiNWs forests for 24 hours in PBS (bar: 200 μm).

FIG. 12A through FIG. 12E depict the capture of viruses inside thepSiNWs microfluidic devices. FIG. 12A: Control group: without injectingviruses (bar: 200 μm). FIG. 12B: Capture of green immunofluorescentstained avian influenza virus (bar: 200 μm). FIG. 12C: SEM image of acaptured virus (scale bar: 150 nm). FIG. 12D: Virus fluorescenceintensity measured under different flow rates. FIG. 12E: RT-qPCR ofinflow sample and outflow samples to calculate capture efficiency.

FIG. 13 is a table listing the virus capture efficiency of the device.

FIG. 14A through FIG. 14C depict the results of releasing andpropagation of released virus. FIG. 14A: Releasing virus by degradingpSiNWs forests for 24 hours in PBS (bar: 200 μm). FIG. 14B: RT-qPCR ofinflow sample and recovery samples to calculate recovery efficiency.Dissolved PBS: pSiNWs degradation solution in PBS without viruses. FIG.14C: HA test on samples after released virus and propagation inembryonated chicken eggs. PBS control: pSiNWs degradation solution inPBS without viruses; Positive control: virus solution with a titer 1:2⁹;Negative control: DI water.

FIG. 15A through FIG. 15C depict the collection of released virus byantibody conjugated magnetic beads. FIG. 15A: Sketch of detectingreleased viruses by magnetic beads (Fluorescence of streptavidin isred). FIG. 15B: Released virus. FIG. 15C: Control group. Viruses werecaptured by H5 antibody conjugated magnetic beads and then stained withred fluorescence labeled streptavidin conjugated to biotin labeled H5antibody. The red fluorescence in the released virus solution was muchstronger than that of the control group, indicating there were manyviruses released in the solution and collected at the outlet of thedevice.

FIG. 16 is a table listing the virus recovery efficiency of the device.

FIG. 17, comprising FIG. 17A and FIG. 17B, depicts the design andworking principle of the vertically-aligned carbon nanotube (VACNT)herringbone device for virus enrichment and diagnosis. (FIG. 17A)Illustration of virus diagnosis procedures inside the device, include 1)size-based virus isolation and enrichment, 2) on-chip virus lysis forgenetic detection or on-chip immunofluorescence assay, 3)culture/propagation of isolated AIV in fertilized chicken eggs. (FIG.17B) Assembled device image and porous herringbone structure under EMincluding optical (i), SEM (ii, iii, iv), and TEM (v) images of VACNTforest in herringbone structure.

FIG. 18, comprising FIG. 18A through FIG. 18C, depicts the results ofexperiments demonstrating VACNT synthesis morphology andcharacterization after 30 minutes chemical vapor deposition (CVD)synthesis. (FIG. 18A) Cross-sectional SEM images of VACNT growing on 4nm, 7 nm, and 10 nm thick iron catalyst layer. (FIG. 18B)Characterization of CNT diameter. (FIG. 18C) Characterization of CNTdensity.

FIG. 19, comprising FIG. 19A through FIG. 19C, depicts the results of agap size measurement with capture efficiency experiment, wherein gapsize refers to the average distance between each CNT. (FIG. 19A)Calculated VACNT forest gap size. (FIG. 19B) Capture efficiency of 95 nmgap size device characterized by fluorescent nanospheres. (FIG. 19C)Top-viewed fluorescence images of herringbone structure after applied amixture of particles with diameters of 20 nm (red), 100 nm (green), and1000 nm (blue).

FIG. 20, comprising FIG. 20A through FIG. 20D, depicts the results ofexperiments demonstrating the capture and on-chip detection of Avianinfluenza virus H5N2 from a swab sample. (FIG. 20A) Histogram of H5N2diameter with a TEM image (inset). (FIG. 20B) On-chip indirectimmunofluorescence detection. (FIG. 20C) SEM images of H5N2 trappedinside the herringbone structure. (FIG. 20D) DNA gel electrophoresis forgenetic analysis after on-chip lysis.

FIG. 21 depicts RT-qPCR demonstration of H5N2 enrichment and improvementof detection limit from 1ELD₅₀ to 0.1ELD₅₀.

FIG. 22A and FIG. 22B depict the working principle of virus enrichmentand concentration from field samples. (FIG. 22A) A field samplecontaining viruses (purple spheres) is collected by a cotton swab or asa tissue sample. (FIG. 22B) The supernatant of the field sample flowsthrough the CNT-STEM and the viruses are enriched within the device.Inset (right): illustration of size-based virus enrichment by thealigned N-MWCNTs. Inset (bottom right): SEM image (scale bar, 100 nm) ofthe H5N2 AIV virions trapped inside the aligned N-MWCNTs. Inset (bottomleft): dark field TEM image (scale bar, 100 nm) of enriched H5N2 AIVafter the aligned N-MWCNTs structures were retrieved from the CNT-STEM.

FIG. 23A and FIG. 23B depict the fabrication process and the testingsetup of the CNT-STEM. (FIG. 23A) Fabrication process of CNT-STEM. (i)Iron catalyst thin film was deposited on a prime silicon wafer andpatterned by a lift-off process. (ii) The aligned CNT was selectivelysynthesized on patterned silicon surface during AACVD. (iii) CNT-STEMwas formed by bonding a PDMS chamber with fluidic access to siliconsubstrate. Arrows label sample flow direction from the inlet to theoutlet. (FIG. 23B) Top and side view of the testing setup. Thevirus-containing sample was first filtered through a membrane filter of0.2 μm pore size (not shown here), then loaded into the sample reservoirat the inlet and processed through CNT-STEM via a vacuum sourceconnected through a waste trap at the outlet. The vacuum pressure wasmeasured by a miniature pressure sensor and regulated by a precisionmechanical regulator. Inset shows the CNT-STEM device, scale bar: 1 cm.

FIG. 24 depicts aerosol assisted chemical vapor deposition (AACVD) fornitrogen-doped carbon nanotube (N-MWCNT) synthesis.

FIG. 25 depicts the calculated distance between the iron particles basedon the Delaunay triangle selection algorithm. The inset illustrates thegeometry definition of nearest neighbor particles. The averageparticle-to-particle distance is the mean of r₂ and r₃.

FIG. 26 depicts a diagram of the data processing pipeline for NGS.

FIG. 27A through FIG. 27J depict controlled N-MWCNT growth and tunablesize-based filtration of the CNT-STEM. Microscopy images of the N-MWCNTporous wall in the CNT-STEM (FIG. 27A through FIG. 27E). (FIG. 27A)Bright-field optical microscopy image showing the top view of thedroplet-shaped porous wall made by the aligned N-MWCNTs on a siliconsubstrate. Scale bar, 2 mm. (FIG. 27B) Scanning electron microscopy(SEM) image of the aligned N-MWCNTs. Scale bar, 50 μm. (FIG. 27C)High-magnification SEM image showing a side view of the alignedN-MWCNTs. Scale bar, 100 nm. (FIG. 27D) Transmission electron microscopy(TEM) image of AACVD synthesized N-MWCNTs of various diameters. Scalebar, 20 nm. (FIG. 27E) High-resolution TEM image showing the multiwallstructure N-MWCNT. Scale bar, 5 nm. (FIG. 27F) Formation of ironnanoparticle catalyst and growth of N-MWCNTs on iron catalyst layerswith different thicknesses. Top row: SEM images showing top views ofiron particles formed on a silicon surface after 850° C. thermaltreatment in AACVD. Thicknesses of iron catalyst thin films are 1, 3,6.5, 9 and 12 nm. Middle and bottom row: SEM images of cross-sectionalviews of an aligned N-MWCNT structure after 30 minutes N-MWCNT growth byAACVD. Scale bars, top: 100 nm, middle: 10 μm, bottom: 200 nm. Diameter(FIG. 27G) and density (FIG. 27H) of iron particles (red) and N-MWCNT(black) as a function of iron film thickness (n=8). (FIG. 27I)Inter-tubular distance measured by image analysis as a function of ironfilm thickness (n=8). (FIG. 27J) Calculated porosity of the N-MWCNT wall(n=8).

FIG. 28A through FIG. 28F depict Raman spectra of the newly synthesizedN-MWCNT structures on silicon substrates and the effect of the synthesistime on the height, diameter and density of the aligned N-MWCNTstructure. Geometrical parameters were measured from SEM images. (FIG.28A) Raman spectra of the N-MWCNT structures synthesized on 3 nm, 6.5 nmand 12 nm thick iron catalyst thin films. The Raman spectra indicate thealigned N-MWCNT has D, G and D′ band peaks at 1352, 1578 and 2659 cm⁻¹,respectively. The results are consistent with previous studies onN-MWCNT. (FIG. 28B) Plot of the peak height ratio of the D band and Gband of the N-MWCNT structures formed on 3 nm, 6.5 nm and 12 nm thickiron catalyst thin films over synthesis time. Thicker iron catalystlayer results in lower D/G band ratio. (FIG. 28C) Height of N-MWCNTstructure synthesized for 30 minutes on 1 nm, 3 nm, 6.5 nm, 9 nm and 12nm thick iron thin films (n=8). (b-d) The effect of the synthesis timeon the height (FIG. 28D), diameter (FIG. 28E) and linear density (FIG.28F) of the N-MWCNT structure. The N-MWCNT was grown on 3 nm, 6.5 nm and12 nm thick iron catalyst thin films under 5, 10, 20, 30 and 40 minutesof AACVD synthesis (n=8).

FIG. 29A and FIG. 29B depict the measured particle size-dependentfiltration characteristics of CNT-STEMs with N-MWCNT inter-tubulardistances of 25 nm, 95 nm, and 325 nm, using small molecule fluoresceinand fluorescent polystyrene nanospheres of 20 nm, 50 nm, 100 nm, 140 nm,400 nm, and 1000 nm in diameter. (FIG. 29A) Fluorescence microscopeimages showing fluorescein solution and fluorescent polystyrenenanospheres of various diameters being filtered by the CNT-STEM. Thedirection of the flow is from right to left as indicated by the redarrows. Yellow lines delineate the contours of the N-MWCNT structures.Scale bars, 50 μm. (FIG. 29B) Penetration of fluorescein andfluorescence polystyrene nanospheres through the N-MWCNT structure(n=8).

FIG. 30A and FIG. 30B depict the characterization of size-based particlecapture by CNT-STEM. (FIG. 30A) Diameter distribution of fluorescentpolystyrene nanospheres measured by laser diffraction. (FIG. 30B)Florescence microscopic image showing the transport of 100 nmfluorescently labeled nanospheres in CNT-STEM device with 95 nminter-tubular distance. Inset is a SEM image of nanospheres trappedinside N-MWCNT structure of the CNT-STEM.

FIG. 31 depicts a table listing the measurement of the inter-tubulardistance of N-MWCNT forest and the corresponding critical particle sizesof CNT-STEM.

FIG. 32A through FIG. 32D depict the enrichment and concentration ofvirus swab samples by CNT-STEM. (FIG. 32A) Top-view illustration ofviruses passing through and captured by the N-MWCNT array. (FIG. 32B)On-chip indirect fluorescent antibody (IFA) staining of captured H5N2AIV inside CNT-STEMs with 25 nm, 95 nm, and 325 nm inter-tubulardistances. Fluorescence microscopy images of the CNT-STEMs, red arrowsindicate the flow direction. Yellow dotted lines delineate the contoursof the N-MWCNT structures. The control sample was allantoic fluidwithout viruses. Scale bars, 25 μm. (FIG. 32C) Capture efficiency ofCNT-STEMs with inter-tubular distance of 25, 95, and 325 nm measured byrRT-PCR (n=6). (FIG. 32D) Examples of rRT-PCR AIV detection curves forvirus titers of 10⁴, 10³, 10², 10¹, and 10⁰ EID₅₀/mL without (i) andwith (ii) CNT-STEM enrichment. a.u., arbitrary unit.

FIG. 33 depicts the standard curve for the rRT-PCR detection of H5N2 AIV(n=4 each). The rRT-PCR assay had efficiency of 99.66% with the slope ofthe standard curve −3.33. The concentration of the original H5N2 sample(no dilution) was 1.8×10⁸ EID₅₀. No signal was detected after 10⁷dilution thus the detection limit here was 1.8×10² EID₅₀/mL.

FIG. 34 depicts the capture efficiency measurement of CNT-STEM with 25nm, 95 nm, and 325 nm inter-tubular distances when loading H5N2 AIV of10⁶ EID₅₀/mL titer into each device (n=6). The Ct values of filtratesand the original sample were measured (n=6 each).

FIG. 35 depicts the rRT-PCR curves of H5N2 AIV samples of 10 EID₅₀/mLand 10² EID₅₀/mL titers without enrichment and those of 0.1 EID₅₀/mL and1 EID₅₀/mL titers with CNT-STEM enrichment (n=6).

FIG. 36 depicts the compatibility test of N-MWCNT to rRT-PCR. The“H5N2+N-MWCNT” samples were prepared by scraping N-MWCNT from theCNT-STEM without virus processing using razor blade and then mixed withswab-mimicking H5N2 AIV samples of 5×10⁴ EID₅₀/mL titer for rRT-PCRdetection (n=5).

FIG. 37A through FIG. 37C depict the results of CNT-STEM enrichingviruses viability and improving the minimal virus concentration of virusisolation. (FIG. 37A) Illustration showing inoculation of virus-embeddedN-MWCNT structure into embryonated chicken egg (ECE). (FIG. 37B)Dot-ELISA detection of H5N2 AIV after virus cultivation in ECEs. Virussamples inoculated into chicken eggs were either from original virussamples in the titers of 10², 10³, and 10⁴ EID50/mL or CNT-STEM enrichedsamples of the same corresponding original titers. A darken spot with apositive sign indicated that H5N2 AIV successfully propagated inside thechicken egg. (FIG. 37C) Success rates of AIV isolation via egginoculation with and without using the CNT-STEM for original virustiters of 10⁴, 10³, and 10² EID₅₀/mL (n=10).***: p≤0.001.

FIG. 38A through FIG. 38C show CNT-STEM prepares mimic field samples forNGS virus analysis. (FIG. 38A) Raw reads generated by NGS without (i)and with (ii) CNT-STEM enrichment. (FIG. 38B) Circos plots of assembledcontiguous sequences generated from NGS reads of the CNT-STEM enrichedH5N2 LPAIV samples. Track 1 [outermost]: scale mark. Track 2: iSNV.Track 3: variants comparing to H5N2 AVI strain A/mallard/WI/411/1981.Track 4: coverage (black) on scale of 0 to 30k reads. Track 5: de novoassembled contigs after CNT-STEM enrichment (grey). Track 6: openreading frames (green). Color coding in tracks 2 and 3: deletion(black), transition (A-G: fluorescent green, G-A: dark green, C-T: darkred, T-C: light red), transversion (A-C: brown, C-A: purple, A-T: darkblue, T-A: fluorescent blue, G-T: dark orange, T-G: violet, C-G: yellow,G-C: light violet). (FIG. 38C) Phylogenetic tree plots generated bycomparing the HA (i) and NA (ii) genes of the sequenced H5N2 AIV(highlighted in red) to those of closely related AIV strains isolated inNorth America from Genbank.

FIG. 39 depicts a table listing the assembled contigs of the LP H5N2 AIVsample enriched by CNT-STEM.

FIG. 40A and FIG. 40B depict tables showing the phylogenetic analysis ofthe sequenced H5N2 strain (A/chicken/PA/7659/1985) to closely relatedH5N2 AIV strains isolated from US/Canada in Genbank for (FIG. 40A) theHA gene and (FIG. 40B) the NA gene.

FIG. 41 depicts the results of rRT-PCR detection of the H11N9 AIV duckswab with and without CNT-STEM enrichment.

FIG. 42A and FIG. 42B depict the identification of an emerging AIV H11N9strain from a surveillance swab sample using CNT-STEM followed by NGSand de novo genome sequence assembly. (FIG. 42A) Circos plot ofassembled H11N9 contigs generated by NGS from a CNT-STEM enriched wildduck swab pool. Track 1 [outermost]: scale mark. Track 2: IdentifiediSNV. Track 3: Variants compared to a previously reported H11N9 AIVstrain (A/duck/1VIN/Sg-00118/2007). Track 4: Coverage on scale of 0 to50 reads (black). Track 5: de novo assembled contigs after CNT-STEMenrichment (grey). Track 6: Open reading frames (green). Color coding intracks 2 and 3: deletion (black), transition (A-G: fluorescent green,G-A: dark green, C-T: dark red, T-C: light red), transversion (A-C:brown, C-A: purple, A-T: dark blue, T-A: fluorescent blue, G-T: darkorange, T-G: violet, C-G: yellow, G-C: light violet). (FIG. 42B)Phylogenetic tree plots generated by comparing the HA (a) and NA (b)genes of the sequenced H11N9 AIV (highlighted in red) to selectedclosely related AIV strains isolated in North America from Genbank.

FIG. 43 is a table listing the assembled contigs of the H11N9 AIV fieldsample enriched by CNT-STEM.

FIG. 44A and FIG. 44B depict tables showing the phylogenetic analysis ofthe emerging H11N9 strain (A/duck/PA/02099/2012) to previously reportedand closely related AIV strains for (FIG. 44A) the HA gene and (FIG.44B) the NA gene.

FIG. 45A and FIG. 45B depict the identification of a new IBDV strainfrom a turkey eyelid tissue sample using CNT-STEM followed by NGS and denovo genome sequence assembly. (FIG. 45A) Circos plots of assembledcontiguous sequences generated from NGS reads of the CNT-STEM enrichedIBDV samples. Track 1 [outermost]: scale mark. Track 2: iSNV. Track 3:variants comparing to OH Strain (U30818) (b). Track 4: coverage (black)on scale of 0 to 2625 reads. Track 5: de novo assembled contigs afterCNT-STEM enrichment (grey). Track 6: open reading frames (green). Colorcoding in tracks 2 and 3: deletion (black), transition (A-G: fluorescentgreen, G-A: dark green, C-T: dark red, T-C: light red), transversion(A-C: brown, C-A: purple, A-T: dark blue, T-A: fluorescent blue, G-T:dark orange, T-G: violet, C-G: yellow, G-C: light violet). (FIG. 45B)Phylogenetic tree plots generated by comparing the open reading framesVP2/VP3/VP4 (a) and VP1 (b) of MDV/Turkey/PA/00924/14 (highlighted inred) to previously reported IBDVs.

FIG. 46 is a table comparing contigs of the unknown virus(IBDV/Turkey/PA/00924/14) generated by de novo assembly after CNT-STEMenrichment and NGS to the closest IBDV strains in Genbank.

FIG. 47A and FIG. 47B depict tables showing the SNP/variant analysis ofthe “unknown” virus (IBDV/Turkey/PA/00924/14) to sequenced IDBV virusstrains for (FIG. 47A) capsid proteins VP2, VP3, and VP4 and (FIG. 47B)VP1.

FIG. 48 is a table comparing CNT-STEM to several reportedultrafiltration devices.

FIG. 49 is a table listing the yield and reliability analysis ofCNT-STEM fabrication, assembly and testing.

FIG. 50A is a table comparing the cycle threshold (Ct) values ofenriching plum pox virus using a current USDA virus enrichment protocolversus an enrichment protocol using CNT-STEM with 25 nm inter-tubulardistance.

FIG. 50B is a table listing the results of enriching herpes simplexvirus using CNT-STEM.

DETAILED DESCRIPTION

The present invention provides for an enrichment platform device andmethods for size-based capture of particles in solution. The inventionis useful for label-free capture of any particle. The invention is alsouseful for filtering particles out of solution. The invention is alsouseful for concentrating and isolating viable particles out of solutionfor analysis.

In one embodiment, the device comprises vertically-aligned carbonnanotubes (VACNT). In one embodiment, the device comprises nanowires. Inone embodiment, the enrichment platform device is a microfluidic device.In another embodiment, the enrichment platform device is a portabledevice wherein sample solutions are passed through by hand push.

The invention provides a method for making the enrichment platformdevice. In one embodiment, the method comprises chemical-vapordeposition. In another embodiment, the method comprises deepreactive-ion etching (DRIE), metal assisted silicon etching, and wetetching.

The invention provides methods for capturing particles in solution. Inone embodiment, the method filters particles out of a solution. In oneembodiment, the method captures particles in a solution for analysis. Inone embodiment, the method captures and releases viable particles foranalysis.

Definitions

It is to be understood that the figures and descriptions of the presentinvention have been simplified to illustrate elements that are relevantfor a clear understanding of the present invention, while eliminating,for the purpose of clarity, many other elements found in typical tissueengineering system and methods. Those of ordinary skill in the art mayrecognize that other elements and/or steps are desirable and/or requiredin implementing the present invention. However, because such elementsand steps are well known in the art, and because they do not facilitatea better understanding of the present invention, a discussion of suchelements and steps is not provided herein. The disclosure herein isdirected to all such variations and modifications to such elements andmethods known to those skilled in the art.

Unless defined elsewhere, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are described.

As used herein, each of the following terms has the meaning associatedwith it in this section.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

“About” as used herein when referring to a measurable value such as anamount, a temporal duration, and the like, is meant to encompassvariations of ±20%, ±10%, ±5%, ±1%, and ±0.1% from the specified value,as such variations are appropriate.

The term “carbon nanotubes” (CNTs) is used herein in a generic sense toinclude single-walled and multi-walled carbon nanotubes, carbonnanofibers, carbon nanofilaments, and carbon nanoropes.

The term “channel” refers to a gap between any two protrusions. Thechannels of the present invention may be any convenient size or shape.

As used herein, “chemical vapor deposition” refers to plasma enhancedchemical vapor deposition or thermal chemical vapor deposition.

A “disease” is a state of health of a living organism, wherein theliving organism cannot maintain homeostasis, and wherein if the diseaseis not ameliorated then the living organism's health continues todeteriorate.

In contrast, a “disorder” in a living organism is a state of health inwhich the living organism is able to maintain homeostasis, but in whichthe living organism's state of health is less favorable than it would bein the absence of the disorder. Left untreated, a disorder does notnecessarily cause a further decrease in the living organism's state ofhealth.

A disease or disorder is “alleviated” if the severity of a symptom ofthe disease or disorder, the frequency with which such a symptom isexperienced by a patient, or both, is reduced.

As used herein, the term “doped” means that for any given carbonnanotube, at least a portion of the carbon sites in the graphiticstructure of the carbon nanotube are filled with atoms of the dopingmaterial instead of with carbon atoms, such that the portion of carbonsites so filled with the doping material would be detectable by commonanalytical means known in the art such as, for example, x-rayphotoelectric spectroscopy (XPS).

The term “nanowire” as used herein is meant to describe a nanoscaleparticle typically of high aspect ratio. An “aspect ratio” is the lengthof a first axis of a nanostructure divided by the average of the lengthsof the second and third axes of the nanostructure, where the second andthird axes are the two axes whose lengths are most nearly equal eachother. For example, the aspect ratio for a perfect rod would be thelength of its long axis divided by the diameter of a cross-sectionperpendicular to (normal to) the long axis. Consequently, a nanowire hasan aspect ratio greater than about 1.5 or greater than about 2. Shortnanowires, sometimes referred to as “nanorods,” typically have an aspectratio between about 1.5 and about 10. Longer nanowires may have anaspect ratio greater than about 10, or even greater than about 10,000.The diameter of a nanowire is typically less than about 500 nm and maybe less than 200 nm. In some examples, the diameter of a nanowire mayeven be less than about 5 nm.

The terms “patient,” “subject,” “individual,” and the like are usedinterchangeably herein, and refer to any living organism, or cellsthereof whether in vitro or in situ, amenable to the methods describedherein. In certain non-limiting embodiments, the patient, subject orindividual is a human, an animal, an insect, or a plant.

Throughout this disclosure, various aspects of the invention can bepresented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6, etc., as well as individual numberswithin that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, 6, and anywhole and partial increments therebetween. This applies regardless ofthe breadth of the range.

Enrichment Platform Device

The invention provides enrichment platform devices for size-basedcapture of particles in solution. The enrichment platform device isuseful for label-free capture of any particle.

Referring now to FIG. 1A, one embodiment of a vertically-aligned carbonnanotubes (VACNT) enrichment platform device 10 is depicted. VACNTenrichment platform device 10 comprises substrate 12, VACNT 14, andcover 16. Substrate 12 can be any substrate known in the art, including,but not limited to, silicon, glass, sapphire, metals, polymers, and thelike.

VACNT 14 is attached to substrate 12 at their distal ends. VACNT 14comprises at least one CNT selected from the group including, but notlimited to, single-wall CNT, double-walled CNT, multi-wall CNT, andcombinations thereof. In one embodiment, VACNT 14 is doped. Non-limitingexamples of doped VACNT 14 include nitrogen-doped VACNT, boron-dopedVACNT, silicon-doped VACNT, aluminum-doped VACNT, phosphorus-dopedVACNT, and lithium-doped VACNT. In one embodiment, VACNT 14 is doped toenhance biocompatibility, such that the viability of captured particlesis preserved.

VACNT 14 is arranged in forests, such that a forest comprises aplurality of VACNT 14. Forests of VACNT 14 can be in any suitablearrangement. Non-limiting examples of VACNT forest arrangements includeherringbone pattern (FIG. 17B), continuous sidewalls (FIG. 22B), solidblocks, striped pattern, concentric circles, and the like.

A forest of VACNT 14 comprises gaps between VACNT. In one embodiment,the gap size is at least 1 nm. In various embodiments, the gap size isbetween 1 nm and 500 nm. In one embodiment, the gap size is dimensionedto fit the diameter of the particles to be captured. In variousembodiments, the device comprises a plurality of VACNT forests havingdifferent properties. For example, the plurality of VACNT forests mayhave different gap sizes, different diameters, different thicknesses,and different densities. The device may also comprise a plurality ofVACNT forests having single-wall CNT, double-walled CNT, multi-wall CNT,and combinations thereof. The device may also comprise a plurality ofVACNT forests having differently doped CNT.

The VACNT forests and substrate 12 are enclosed by cover 16. Cover 16may be made from any material, including, but not limited to, plastics,metals, glass, polymers, polydimethylsiloxane (PDMS), and the like.Cover 16 comprises at least one inlet 18 and at least one outlet 20 foringress and egress of sample solution. In one embodiment, cover 16 isbonded to both substrate 12 and VACNT 14, such as in FIG. 1A. In anotherembodiment, cover 16 is bonded to substrate 12 only, such that VACNT aresecured only to substrate 12 at their distal ends and are not connectedto anything at their proximal ends, such as in FIG. 1B. In oneembodiment, cover 16 is removable.

In one embodiment, the VACNT enrichment platform device 30 is amicrofluidic device. In another embodiment, the VACNT enrichmentplatform device 30 is a portable device wherein sample solutions arepassed through by hand push.

Referring now to FIG. 2B, one embodiment of a nanowire enrichmentplatform device 30 is depicted. Nanowire enrichment platform device 30comprises substrate 12, nanowires 32, and cover 34 (FIG. 4). Substrate12 can be any substrate known in the art, including, but not limited to,silicon, glass, metals, polymers, and the like.

Nanowires 32 are attached to substrate 12. Nanowires 32 can be made ofany suitable material, including, but not limited to, silicon, zinc,zinc oxide, nickel, and the like. Further examples include: elementalsemiconductor nanowires such as silicon and germanium; III-V compoundsemiconducting nanowires such as gallium arsenide, indium arsenide, andindium phosphide; II-VI semiconductor nanowires such as zinc sulfide,zinc selenide, cadmium sulfide, and cadmium selenide; metalsemiconductor nanowires such as gold-silicon, and nickel-silicon; metalnanowires such as platinum, gold, aluminum, and iron; oxide nanowiressuch as zinc oxide, magnesium oxide, manganese dioxide, silicon dioxide,and titanium dioxide; sulfide nanowires such as copper sulfide, nickelsulfide, and iron sulfide; alloy nanowires such as cobalt-copper,iron-silver, and cobalt-silver; superconducting nanowires such asniobium nitride and yttrium barium copper oxide; and polymer nanowiressuch as polypyrrole and polyvinylpyrrolidone. In one embodiment,nanowires 32 comprise the same material as substrate 12. In someembodiments, nanowires 32 are uncoated. In other embodiments, nanowires32 are provided with a coating. The nanowire coating can comprise anysuitable coating, including, but not limited to: nickel, copper,silicon, aluminum, zinc, and the like. In one embodiment, the nanowirecoating is enhances biocompatibility, such that the viability ofcaptured particles is preserved.

Nanowires 32 comprise gaps between each nanowire. In one embodiment, thegap size is at least 1 nm. In various embodiments, the gap size isbetween 1 nm and 500 nm. In one embodiment, the gap size is dimensionedto fit the diameter of the particles to be captured. In variousembodiments, the device comprises a plurality of nanowires 32 havingdifferent properties. For example, the nanowires 32 may have differentgap sizes, different diameters, different thicknesses, and differentdensities. The device may also comprise a plurality of nanowirescomprising different materials. The device may also comprise a pluralityof nanowires having different coatings.

Nanowires 32 and substrate 12 are enclosed by cover 34. Cover 34 may bemade from any material, including, but not limited to, plastics, metals,glass, polymers, polydimethylsiloxane (PDMS), and the like. Cover 34comprises at least one inlet 18 and at least one outlet 20 for ingressand egress of sample solution. In one embodiment, cover 34 is bonded tosubstrate 12 only. In another embodiment, cover 34 is bonded tosubstrate 12 and to nanowires 32. In one embodiment, cover 34 isremovable.

In one embodiment, the nanowire enrichment platform device 30 is amicrofluidic device. In another embodiment, the nanowire enrichmentplatform device 30 is a portable device wherein sample solutions arepassed through by hand push.

Methods of Making

The invention provides methods for making the enrichment platformdevice. In one embodiment, the method is for making a VACNT enrichmentplatform device. In another embodiment, the method is for making ananowire enrichment platform device.

In one embodiment, the method of making a VACNT enrichment platformdevice comprises bottom-up synthesis of CNT. An exemplary diagram isshown in FIG. 24. An iron-catalyst thin film is prepared on a substrateby e-beam evaporation and lift-off process and patterned usinglithography. In various embodiments, additional catalyst materials arecontemplated. Non-limiting examples of catalyst materials include nickeland cobalt. The pattern can be any pattern, including, but not limitedto, a porous herringbone pattern, a droplet pattern, a spiral pattern,and the like. The CNT are synthesized through aerosol-assisted chemicalvapor deposition (AACVD). The CNT synthesis method is not limited toAACVD; rather, the CNT synthesis method encompasses any CNT synthesismethod known in the art. In certain embodiments of the method, at leasta portion of the CNT is vertically aligned. In some embodiments, the allof the CNT are vertically aligned.

The method is amenable to making a plurality of CNT types, including,but not limited to: single-wall CNT, double-walled CNT, multi-wall CNT,and combinations thereof. In one embodiment, the CNT are doped. The CNTcan be doped using any method known in the art, using any suitablematerial known in the art. For instance, non-limiting examples ofmaterials the CNT can be doped with include nitrogen, boron, silicon,aluminum, phosphorus, and lithium. In one embodiment, the CNT are dopedto enhance biocompatibility for maintaining the viability of capturedparticles.

In one embodiment, the gap size between the CNT is controlled byadjusting the thickness of the iron-catalyst layer. For instance, in oneembodiment, increasing the thickness of the iron-catalyst layercorrespondingly increases CNT gap size. In one embodiment, the CNT gapsize is tunable to be in the range of 1-500 nm. In one embodiment, thediameter of the CNT is controlled by adjusting the thickness of theiron-catalyst layer. For instance, in one embodiment, increasing thethickness of the iron-catalyst layer correspondingly increases CNTdiameter. In one embodiment, the density of the CNT is controlled byadjusting the thickness of the iron-catalyst layer. For instance, in oneembodiment, increasing the thickness of the iron-catalyst layercorrespondingly decreases CNT density.

The CNT are encased in a microfluidic device by bonding with a coverhaving at least one inlet and at least one outlet (FIG. 23A). In oneembodiment, the cover is reversibly bonded. The cover may be made fromany material, including, but not limited to, plastics, metals, glass,polymers, polydimethylsiloxane (PDMS), and the like.

In one embodiment, the method of making a nanowire enrichment platformdevice comprises deep reactive-ion etching (DRIE). An exemplary diagramis shown in FIG. 4. A thin photoresist layer is deposited on asubstrate, such as silicon, and patterned using lithography. DRIE isused to create channels in the substrate where photoresist is notpresent. A thicker photoresist layer is deposited over existingphotoresist, and silver nanoparticles (SNP) deposited. Silicon etchingis performed by reacting with the SNP as a catalyst to form poroussilicon nanowires (PSNW). In various embodiments, methods of etchingcommonly used in the art are also contemplated, including metal assistedsilicon etching and wet etching.

In one embodiment, the gap size between the PSNW is controlled byadjusting SNP deposition time. For instance, in one embodiment,increasing SNP deposition time increases SNP size, which correspondinglyincreases PSNW gap size.

The SNP and photoresist is removed and the PSNW encased in amicrofluidic device by bonding with a cover having at least one inletand at least one outlet. In one embodiment, the cover is reversiblybonded. The cover may be made from any material, including, but notlimited to, plastics, metals, glass, polymers, polydimethylsiloxane(PDMS), and the like.

Methods of Use

The invention provides methods of using the enrichment platform devicefor filtering particles, capturing particles, concentrating particles,and releasing viable particles. In one embodiment, the method removesparticles from solution. In one embodiment, the method capturesparticles for analysis. In one embodiment, the method captures andreleases viable particles for analysis.

In one embodiment, the invention provides a method of using theenrichment platform device to filter particles from solution. The gapsize of the enrichment platform device can be tuned to capture particlesthat conventional filters cannot remove, such as in a water filter. Suchparticles can include viruses, bacteria, nanoparticles, nanobeads,nanoshards, and the like.

In one embodiment, the invention provides a method of using theenrichment platform to capture particles for analysis. The gap size ofthe enrichment platform device can be tuned to a specific size for thepurpose of capturing known particles. For instance, if a virus has aknown size range, the gap size of the enrichment platform can be tunedto capture particles of the known size. Passing a solution through theenrichment platform device will enable users to determine whether thesolution contains particles of the known size. The particles can then beanalyzed on the device for purposes such as identification, diagnosis,quantification, and the like. In one embodiment, the method uses aportable enrichment platform device. In one embodiment, the method usessample volumes in the milliliter range. In one embodiment, the samplesolutions are patient-derived solutions, including, but not limited to,blood, urine, saliva, and the like. In certain embodiments, the samplecan comprise a solid such as tissue or stool. In certain embodiments,the sample can comprise a gas, such as a patient's breath, to captureair-borne pathogens. In various embodiments, the sample can be obtainedfrom non-human sources, such as animals, plants, and insects.

In one embodiment, the invention provides a method of using theenrichment platform to capture and release particles while maintainingthe viability of the particles. Particles can include, but are notlimited to, viruses (such as plant viruses, human viruses, herpes, zika,hepatitis C, ebola), microorganisms and parasites (such as bacteria,amoeba, and plasmodium), and their various life stages. The gap size ofthe enrichment platform device can be tuned to a specific size for thepurpose of capturing known particles. For instance, if a particle ofinterest has a known size range, the gap size of the enrichment platformcan be tuned to capture particles of the known size range, such as inFIG. 22B. After a solution is passed through the enrichment platformdevice, the device cover is removed and the captured particles arereleased. The particles can be released by any method known in the art,including, but not limited to, scratching the device surface anddegrading the device nanostructures.

By capturing particles according to size, the enrichment platform devicealso concentrates the population of captured particles. In someembodiments, the methods only require milliliters of sample solution tocapture and isolate a volume of highly concentrated particles. Themethods of the invention are also useful in providing concentratedparticle samples to improve the performance of conventional detectionschemes, including, but not limited to, RT-qPCR, next generationsequencing, and culture.

EXPERIMENTAL EXAMPLES

The invention is further described in detail by reference to thefollowing experimental examples. These examples are provided forpurposes of illustration only, and are not intended to be limitingunless otherwise specified. Thus, the invention should in no way beconstrued as being limited to the following examples, but rather, shouldbe construed to encompass any and all variations which become evident asa result of the teaching provided herein.

Without further description, it is believed that one of ordinary skillin the art can, using the preceding description and the followingillustrative examples, make and utilize the compounds of the presentinvention and practice the claimed methods. The following workingexamples therefore, specifically point out the preferred embodiments ofthe present invention, and are not to be construed as limiting in anyway the remainder of the disclosure.

Example 1: A Porous Silicon Nanowire Forest Based MicrofluidicPoint-of-Care Device for Label-Free Isolation and Release of Viruses

Viruses are infectious nanoscale agents which can infect all types oflife forms, including animals, plants and bacteria. The spread of viralinfections could have a significant negative impact on global health andeconomy (Binder, S. et al., Science 1999, 284(5418):1311-1313; Morens,D. M. et al., PLoS Pathog 2013, 9(7):e1003467; Lederberg, J. et al.,Microbial Threats to Health: Emergence, Detection, and Response.National Academies Press: 2003). They have caused some of the deadliestpandemics in recorded human history, including the 1918 influenzapandemic with an estimated 50 million deaths, the ongoing HIV/AIDSepidemic with 36 million deaths so far (Fauci, A. S. et al., New EnglandJournal of Medicine 2012, 366(5):454-461), and the most recent Ebolaoutbreak resulting in 11306 deaths and 28196 reported cases as ofSeptember 2015 (W. H. O., Situation summary 2015, 3). Moreover,adaptation and changes in viruses, human demographics and behavior,environmental changes, technology and economic development,international travels, and global trades facilitate the rapidinternational spread of viral infections (Khan, K. et al., New EnglandJournal of Medicine 2009, 361(2):212-214; Wilson, M. E., Journal ofapplied microbiology 2003, 94:1-11). Therefore, there is an urgent needfor the development of techniques that can rapidly detect viruses andperform the surveillance of viral diseases almost anywhere.

A number of methods have been developed for viral detection, and theviral antigens, nucleic acids and serological antibodies are the corerepertoire of techniques used for laboratory diagnosis of viralinfections (Herring, A. J. et al., Journal of Clinical Microbiology1982, 16(3):473-477; Lee, Y.-F. et al., Biosensors and Bioelectronics2009, 25(4):745-752; Leland, D. S. et al., Clinical Microbiology Reviews2007, 20(1):49-78; Yeh, Y.-T. et al., Annals of Biomedical Engineering2014, 42(11):2333-2343). The detection of virus-specific IgM antibodiesallows a diagnosis to be made from a single specimen and is uniquelyuseful for defining specific antiviral immunity. However, serology isfrequently subject to high levels of off-target cross-reactions and mayoverlook acute infections as the immune system takes several weeks toproduce relevant IgM antibodies. In contrast, the detections on thebasis of viral nucleic acids and antigen can directly identify virusesin specimen allowing prompt diagnosis and emergency treatment, oftenwithin the same day, although the isolation and enrichment of viruses isa great challenge in the set-up given the extremely low virus levels inthe early stage of viral infections. Generally, the virus isolationmethods fall into two categories: biological methods and physicalmethods (Lee, Y.-F. et al., Biosensors and Bioelectronics 2009,25(4):745-752; Kim, Y.-G. et al., Biosensors and Bioelectronics 2009,25(1):253-258; Tam, P. D. et al., Journal of Immunological Methods 2009,350(1-2):118-124). Biological methods use bioaffinities betweenantibodies and virus surface antigen to isolate viruses, in which theexpression of known antigen and the availability of relevant antibodiesmust be simultaneously satisfied. These methods might face pitfalls indealing with unknown or unidentified viruses. In addition, thedetachment of isolated virus from antibodies functionalized surfacewhile keeping viruses intact for subsequent analysis or culture posesanother significant challenge. For example, after isolation the entirevirus-antibodies complex rarely can infect living cells. Moreover,biological methods are usually labor intensive and require experiencedpersonnel. The technical challenges in surface functionalization,including heterogeneous conjugation of antibodies and surfacedenaturation, also need to be addressed. In comparison, physicalmethods, including differential ultracentrifugation, dielectrophoresis,and filtration, mainly exploit differences in density, electricalaffinity, and size between viruses and impurities (Reimer, C. B. et al.,Science 1966, 152(3727):1379-1381; Collins, J. E. et al., Journal ofVeterinary Diagnostic Investigation 1992, 4(2):117-126; Benfield, D. A.et al., Journal of Veterinary Diagnostic Investigation 1992,4(2):127-133; Morgan, H. et al., Biophysical Journal 1999,77(1):516-525; Green, N. G. et al., Journal of Biochemical andBiophysical Methods 1997, 35(2):89-102). They are label-free andvirus-friendly, offering excellent flexibility in subsequent molecularanalysis. Among these methods, size-based filtration is frequently usedas most viruses have very unique size distribution spectrums, rangingfrom 20 nm to 400 nm (FIG. 2A) (Collins, J. E. et al., Journal ofVeterinary Diagnostic Investigation 1992, 4(2):117-126; Benfield, D. A.et al., Journal of Veterinary Diagnostic Investigation 1992,4(2):127-133; Yeh, Y. T. et al., Micro Electro Mechanical Systems(MEMS), IEEE 26th International Conference 20-24 Jan. 2013; pp 951-954;Patolsky, F. et al., Nat. Protocols 2006, 1(4):1711-1724; Yeh, Y. T. etal., SENSORS, IEEE, 1-4 Nov. 2015; pp 1-4; Xia, Y. et al., 18thInternational Conference on Solid-State Sensors, Actuators andMicrosystems (TRANSDUCERS), 21-25 Jun. 2015; pp 444-447; Chen, G. D. etal., Small 2011, 7(8):1061-1067; Yeh, Y. T. et al., 18th InternationalConference on Solid-State Sensors, Actuators and Microsystems(TRANSDUCERS), 21-25 Jun. 2015; pp 747-750). Therefore, viruses can bedifferentiated from impurities, such as proteins, bacteria and mammaliancells, allowing size-based isolation. Additionally, in practicalapplications, the gap distance of a filter can be tuned to obstructdifferent viruses with varying sizes. Carbon nanotubes (CNT) and siliconnanowires have attracted special attention. Their light weight, hightensile strength, biocompatibility, tunable gap distance, and reliableproduction are particularly useful for isolating viruses by size (Yeh,Y. T. et al., Micro Electro Mechanical Systems (MEMS), IEEE 26thInternational Conference 20-24 Jan. 2013; pp 951-954; Patolsky, F. etal., Nat. Protocols 2006, 1(4):1711-1724; Yeh, Y. T. et al., SENSORS,IEEE, 1-4 Nov. 2015; pp 1-4; Xia, Y. et al., 18th InternationalConference on Solid-State Sensors, Actuators and Microsystems(TRANSDUCERS), 21-25 Jun. 2015; pp 444-447; Chen, G. D. et al., Small2011, 7(8):1061-1067; Yeh, Y. T. et al., 18th International Conferenceon Solid-State Sensors, Actuators and Microsystems (TRANSDUCERS), 21-25Jun. 2015; pp 747-750; Wang, Z. et al., Lab on a Chip 2013,13(15):2879-2882; Zhang, G.-J. et al., Analytica Chimica Acta 2012,749:1-15). Recently, it was successfully demonstrated that the CNTforests with tunable gap distance can be used as filters to isolateviruses for propagation and sequencing (Yeh, Y. T. et al., 18thInternational Conference on Solid-State Sensors, Actuators andMicrosystems (TRANSDUCERS), 21-25 Jun. 2015; pp 747-750). Siliconnanowires were frequently used in field effect transistors for labelfree viral detection (Patolsky, F. et al., Nat. Protocols 2006,1(4):1711-1724; Zhang, G.-J. et al., Sensors and Actuators B: Chemical2010, 146(1):138-144; Chen, K.-I. et al., Nano Today 2011,6(2):131-154), but have never been explored for viral isolation.

The following study reports a novel porous silicon nanowires (pSiNWs)forest-based microfluidic point-of-care (POC) device for isolating andreleasing viruses. The POC device has the dimensions 22 mm×10 mm×3 mm.The pSiNWs forest with well controlled inter-wire space was prepared bymetal-assisted wet etching within curved channels (FIG. 2B). In such adesign, the curved channels introduce the circulation in the plane ofthe cross-section (Di Carlo, D. et al., Proceedings of the NationalAcademy of Sciences 2007, 104(48):18892-18897; Hou, H. W. et al.,Scientific Reports 2013, 3:1259; Karabacak, N. M. et al., Nat. Protocols2014, 9(3):694-710), which can spontaneously bring viruses andimpurities to the pSiNWs forest. Later, viruses in a certain size rangecan pass through the gaps and physically trapped inside the forest.Meanwhile, larger impurities can be directly excluded, and smaller onescan escape from forest in continuous fluidic flow. To further improvethe isolation efficiency, the curvature and dimensions of channels werejudiciously optimized, allowing this new POC device to efficientlyfiltrate viruses or nanoparticles in similar size. The results show thatapproximately 50% of influenza viruses can be isolated in 30 minutes.Moreover, the pSiNWs forest is biodegradable in physiological conditionsattributed to the extensive porous surfaces (Anderson, S. H. C. et al.,physica status solidi (a) 2003, 197(2):331-335; Chiappini, C. et al.,Advanced Functional Materials 2010, 20(14):2231-2239), enabling therelease and harvest of trapped viruses in 24 hours for further cultureand molecular analysis. Together, combined with the portability, highisolation efficiency, large sample capacity, and unique viral releasemechanism, this POC devices can provide much faster access to results ator near the sites of the patient care, discover unknown virus, andmonitor infectious diseases.

The materials and methods are now described.

Materials

4 inch <1 0 0> prime silicon wafer with resistivity 0.001-0.005 Ω·cm(University Wafer, Mass., USA). Positive photoresist SPR 3012, positivephotoresist AZ P4620, 49% hydrofluoric acid, and 30% hydrogen peroxide(Penn State University Nanofab). 0.1 N silver nitrate solution (AcrosOrganics). 75 nm Fluoro-Max Dyed Green Aqueous Fluorescent Particles,400 nm Fluoro-Max Dyed Green Aqueous Fluorescent Particles, goatanti-Mouse IgG (H+L) secondary antibody Alexa Fluor 488 conjugate(Thermo Scientific). Cy3-Streptavidin (Invitrogen). Bovine serum albumin(BSA) (Sigma). Polydimethylsiloxane (PDMS) (Dow Corning). 1×PBS (VWR).Filters with 450 nm (Celltreat scientific products) and 200 nm pore size(VWR). Qiagen Onestep RT-PCR Kit (Qiagen, Valencia, Calif.). RNaseinhibitor murine (New England Biolabs. Inc., Mass., USA). Primers forRT-qPCR. H5N2 avian influenza virus and Mouse anti-H5 antibody. 9-11 dayold special-pathogen free embryonated chicken egg.

Fabrication of pSiNWs Forest-Embedded Microfluidic Devices

The fabrication process is illustrated in FIG. 4. A layer of positivephotoresist SPR 3012 in 1.2 μm thickness was spin-coated on a siliconwafer with resistivity of 0.001-0.005 Ω·cm. Curved channels in 100 μmwidth with 100 μm internal curvature radius were patterned on thephotoresist by photolithography followed by DRIE (Alcatel Speeder 100Si)to generate channels in various depth ranging from 20 to 60 μm.Afterwards, SPR 3012 was completely removed, and a layer of positivephotoresist AZ P4620 in 15 μm thickness serving as a protective mask forwet etching was spin-coated and patterned (FIG. 5A and FIG. 5B). pSiNWsforest were prepared within channels by metal-assisted etching followingthe same protocol described previously (Hochbaum, A. I. et al., NanoLetters 2009, 9(10):3550-3554; Qu, Y. et al., Nano Letters 2009,9(12):4539-4543). In brief, the wafer bearing channel patterns wasimmersed into the 0.085% AgNO₃ containing 9.8% HF for 10 to 90 sec atroom temperature (RT) allowing deposition of silver nanoparticles (SNPs)to the bottom and sidewalls of the channels (Equation 1). Then, thewafer was transferred to the etchant bath containing 0.35% H₂O₂ and 9.8%HF for an hours at RT to prepare pSiNWS forests (Equation 2) (Wang, Z.et al., Lab on a Chip 2013, 13(15):2879-2882; Chiappini, C. et al.,Advanced Functional Materials 2010, 20(14):2231-2239; Mohammad, Z. etal., Journal of Micromechanics and Microengineering 2011, 21(6):065006;Shiu, S.-C. et al., Applied Surface Science 2011, 257(6):1829-1834).Finally, after removing SNPs and photoresist AZ P4620 individual deviceswere diced from wafer and firmly bonded with a PDMS cover. (FIG. 2C).4Ag⁺+Si+6F⁻→4Ag+[SiF₆]²⁻  (Eqn. 1)2Ag+H₂O₂+2H⁺→2Ag⁺+2H₂O  (Eqn. 2)Simulation of Flows in the Channels

A finite element method (FEM) model was established to simulate thefluidic field inside the device by the software COMSOL. Laminar flowstationary study was chosen. A meandering channel with five periodic Sshapes (FIG. 6A, FIG. 7A, FIG. 8A) was constructed to study the flowcondition. In the inlet of the channel, the inflow condition was anormal inflow with a flow rate of 1.33 μl/min (8 μl/min in the devicewith 6 channels). In the outlet, the outflow conditions of normal flowand suppress backflow were set up. No slip condition was built on thewalls. A physics-controlled finer mesh was chosen to produce meshes inthe model. The results of velocities in x, y, z direction (FIG. 6Athrough FIG. 6D, FIG. 7A through FIG. 7D, FIG. 8A through FIG. 8D) wereoutput to gain the streamlines in the cross section area of the channel.Capture efficiency (Equation 3) with different channel heights was alsocalculated by comparing fluorescence intensity of the mixture beforethey were injected into channels and after they were gathered in theoutlet, respectively.

$\begin{matrix}{{{Capture}\mspace{14mu}{efficiency}\mspace{14mu}{of}\mspace{14mu}{nano}\mspace{14mu}{beads}} = {1 - {\frac{{FI}_{O}}{{FI}_{I}} \times \frac{V_{O}}{V_{I}}}}} & \left( {{Eqn}.\mspace{14mu} 3} \right)\end{matrix}$FI is the fluorescent intensity of nanobeads solution measured by themicroplate. V is the volume of nanobeads. The subunits I, O, R areinflow, outflow and recovery solution, respectively.Testing Capture Efficiency and Degradation During Device Optimization

All fluorescent images were taken by an Olympus IX 71 microscopeequipped with a digital CMOS camera (C11440, Hamamatsu Photonics,Japan). Before injecting nanobeads or H5N2 virus solution, 1% BSA in1×PBS was flowed through the device at 100 μl/min for 1 hour to preventthe non-specific absorption of nanobeads or virus in the channels. Whentesting the device with nanobeads, a 40 μl mixture of 75 nm and 400 nmnanobeads (the concentrations of both nanobeads in mixture were 375μg/ml, blocked with 1% BSA) was injected into the device, followed byflowing through 210 μl DI water to wash channels. The outflow solution(containing both 40 μl beads mixture and 210 μl DI water) was gatheredin the well of a 96 well plate to calculate the capture efficiency ofnanobeads (Equation 3). To recover captured nanobeads, PBS solutioncontinuously flow through the device for 24, 48, 72 hours at RT.

Testing Capture and Recovery Efficiency of H5N2 Virus

The effect of flow rate on capture efficiency of H5N2 virus wasoptimized first. 40 μl H5N2 virus solution was injected into the deviceat various flow rates ranging from 2 μl/min to 16 μl/min. After rinsingall channels with 1×PBS thoroughly, captured viruses were stained withprimary antibodies (mouse IgG) targeting the H5 hemagglutinin on thevirus surface and the secondary Alexa 488 labeled anti-mouse antibodiesat RT for 45 min, respectively. After washing with 1×PBS thrice, threepositions in the device were randomly selected for measurement offluorescent intensity. To quantify capture efficiency of H5N2 virus, CTvalue of RT-qPCR was used to measure and compare the concentration ofvirus before injecting the device and after flowing through the device(Equation 4). To recover captured viruses, 1×PBS solution continuouslyflew through the device at RT for 24 hours. The recovery solution wasgathered into a 1.5 ml centrifuge tube placing in an ice box. Theconcentration of recovered virus solution was compared to that of inflowvirus solution to calculate the recovery efficiency (Equation 5).

$\begin{matrix}{{{Capture}\mspace{14mu}{efficiency}\mspace{14mu}{of}\mspace{14mu}{virus}} = {1 - {2^{{CT}_{I} - {CT}_{O}} \times \frac{V_{O}}{V_{I}}}}} & \left( {{Eqn}.\mspace{14mu} 4} \right) \\{{{Recovery}\mspace{14mu}{efficiency}\mspace{14mu}{of}\mspace{14mu}{virus}} = {2^{{CT}_{I} - {CT}_{R}} \times \frac{V_{R}}{V_{I}}}} & \left( {{Eqn}.\mspace{14mu} 5} \right)\end{matrix}$V is the volume of virus solution. CT is the CT value of the virussolution by RT-qPCR. The subunits I, O, R are inflow solution, outflowsolution and recovery solution, respectively.Real Time q-RCR

RT-qPCR of H5N2 influenza virus was conducted in a 25 μl reaction systemby one-step RT-qPCR kit (Spackman, E. et al., Journal of ClinicalMicrobiology 2002, 40(9):3256-3260). The primers and probe specific toH5 subtype were used. The reaction mixture contains 5 μl 5× reactionbuffer, 1 μl each of two primers (10 pmol/μl) and probe (5 pmol/μl), 1μl dNTP mixture (10 mM each dNTP), 0.8 μl enzyme mixture, 2 μl RNAtemplate, 13.7 μl RNase-free water and 0.5 μl RNase inhibitor (40 U/μl).The amplification and detection was performed in the 7300 Real time PCRsystem (Applied Biosystem Inc., Foster City, Calif., USA). The thermalcycling profile of RT-qPCR was 50° C. for 30 minutes, 94° C. for 15minutes and 45 cycles of denaturation at 95° C. for 10 seconds andannealing and elongation at 60° C. for 1 minutes. The data was collectedand analyzed by 7300 real time PCR system software (7300 V1.4.0, AppliedBiosystem Inc.) The cycle threshold (CT) value of each sample wascalculated and compared to gain the capture and recovery efficiencies.

Propagation in Embryonated Chicken Egg and Hemagglutination Assay

200 μl released H5N2 influenza virus solution was inoculated andpropagated in 9-11 day old special-pathogen free embroyonated chickenegg. The inoculated eggs were placed inside the incubator for 48 hours.Then the top of the egg was cracked open and the shell was peeledwithout breaking the shell membrane. Allantoic fluid was collected by a3 ml sterile syringe with a 25 G ⅝″ needle. After centrifugation at 8000rpm for 5 minutes, the supernatant containing propagated virus wasfiltered through 450 nm and 200 nm pore sizes filters sequentially. Thehemagglutination assay (HA) was used to test infectious ability ofrecovered viruses. HA test was prepared with 0.5% chicken red bloodcells (RBCs) (Hirst, G. K., The Journal of Experimental Medicine 1942,75(1):49-64). The propagated virus solution was diluted into 2-foldserial and 50 μl of each dilution was added into different wells of a 96well plate. Then, 50 μl 0.5% chicken RBCs was added into each well andincubated in 37° C. for 30 minutes. In the HA test, a negative resultwithout virus appeared as a red dot in the center of the well bottom asthe virus concentration was too low for RBC to settle. A positive resultformed a uniform reddish suspension in the well. The concentration ofvirus was estimated by the dilution times of which the RBCs started tosettle down.

The results are now described.

Fabrication of pSiNWs Forest-Based Microfluidic Devices

For label-free size-based isolation of nanoparticles the inter-wirespacing between pSiNWs should be slightly larger allowing nanoparticlestrapping within the pSiNWs forest. Therefore, the ideal inter-wisespacing was determined first. In metal-assist etching, SNPs server ascatalyst and only silicon beneath SNPs can be effectively etched (FIG.3A) indicating the size of SNPs after deposition directly determine theinter-wire spacing (FIG. 3B). In experiments, pSiNWs forest bearinginter-wire spacing was created on both sidewalls and bottom of channels(FIG. 3C, FIG. 3D), offering large surface area for isolatingnanoparticles. In addition, after 1 hour HF etching at RT, the length ofnanowires was ˜10 μm (FIG. 3E). Mesopores in several nanometers wereobserved on each single nanowires (FIG. 3E). It was speculated thatsilver ions diffusing out the original SNPs can nucleate on nanowires ata certain concentration, extract electrons from the silicon nanowires,form new tiny SNPs, and catalyze the etching along the lateral directionof the nanowires (Qu, Y. et al., Nanoscale 2011, 3(10):4060-4068). Itwas found that by adjusting the deposition time ranging from 45 s to 90s, the average size of SNPs clusters increased from approximately 75nm×100 nm to 140 nm×190 nm (FIG. 3B, FIG. 3F). Accordingly, after wetetching the average size of inter-wire spacing increased from about 180nm×230 nm to 250 nm×330 nm (FIG. 3C, FIG. 3F). As the size of influenzaA viruses used in this study were approximately 80 to 120 nm (Lamb, R.A. et al., Annual Review of Biochemistry 1983, 52(1):467-506), theinter-wire spacing had to be kept slightly larger than 120 nm whichwould require 45 s to 60 s for deposition of SNPs (FIG. 3F). Todetermine the ideal deposition time, 50 inter-wire spacing were randomlyselected and measured in respective group (FIG. 3G, FIG. 3H). Theresults clearly show over 30% of the inter-wire spacing is smaller than120 nm if the deposition time is shortened to 45 s. In contrast, with 60s deposition, almost all inter-wire spacing is larger than the thresholdvalue (FIG. 3H), and the average inter-wire spacing is about 227 nm×291nm (FIG. 3G). Such spacing distribution would allow viruses to enterinto pSiNWs forest and trap them within the forest. On the other hand,microparticles, such as bacteria and platelets, are unable to cross therelatively narrow gap or deeply enter into pSiNWs forest. On thecontrary, other nanoparticles, e.g. serum proteins, are much smaller andcan easily enter and escape from the forest. Thus, deposition time of 60seconds was used to prepare pSiNWs forest for isolating virus influenzaA.

Optimization of Performance of pSiNWs Embedded Microfluidic DevicesUsing Nanobeads

In flow through curved channel geometries, curvature amplifies a lateralinstability that drives a secondary cross-sectional flow field, known asDean flow, characterized by the presence of two counter-rotatingvortices located above and below the horizontal plane of symmetry of thechannel (Di Carlo, D., Lab on a Chip 2009, 9(21):3038-3046). Inertialfocusing of spherical microparticles with diameters ranging between 5and 20 μm has demonstrated the promise of efficient separation as wellas increased throughput (Di Carlo, D. et al., Proceedings of theNational Academy of Sciences 2007, 104(48):18892-18897; Hou, H. W. etal., Scientific Reports 2013, 3:1259; Karabacak, N. M. et al., Nat.Protocols 2014, 9(3):694-710). To date, the Dean flow-based techniquehas not been reported with nanoparticles. In the present design, virusesare expected to be brought into pSiNWs forest by vortices generated inthe channels, and thus the odds of trapping viruses inside the forestcan be improved. Moreover, in the flow the wall lift force pushingviruses against walls was smaller than the opposite Dean force inseveral orders of magnitude (FIG. 9) (Di Carlo, D., Lab on a Chip 2009,9(21):3038-3046; Matas, J.-P. et al., Journal of Fluid Mechanics 2009,621:59-67; Martel, J. M. et al., Scientific Reports 2013, 3:3340). Itwill also guide viruses through inter-wise spacing to pSiNWs forest, andtrap them inside. Given that the curvature of channels is fixed, it washypothesized that vortices that can efficiently bring viruses to pSiNWsforest could be generated by optimizing channel height and flow rate. Tostudy the effect of channel height on Dean flow in the cross-sectionalplane, a FEM model was established to simulate the fluidic field insidethe channel. It was found that the intensity (FIG. 6A through FIG. 6D,FIG. 7A through FIG. 7D, FIG. 8A through FIG. 8D) and position of localvortices (FIG. 10A through FIG. 10C) significantly changed in threegroups. Relatively more local vortices near the channel wall wereobserved in the curved channels with 20 μm height in comparison withthat in the rest of the two groups, and thus it was speculated that moreviruses would be brought into the pSiNWs forest in channels withrelatively lower height. Further, the height effect on captureefficiency (Equation 3) was experimentally investigated usingfluorescence labeled nanobeads. Green or blue fluorescence labelednanobeads in 75 nm and 400 nm sizes mimicking influenza A viruses andimpurities in body fluid, respectively, were used for testing. When thechannel height decreased from 60 μm to 20 μm, at 8 μl/min the captureefficiency of 75 nm nanobeads increased from 6.4% to 14.1%, while thatof the 400 nm nanobeads was approximately 4% in all groups (FIG. 10D).Together, the height optimized from experiments was consistent withsimulation outcome, and thus channels with 20 μm height were preparedfor the following optimization.

To study capture specificity, a 40 μl mixture of nanobeads in 75 nm and400 nm size, respectively, were injected into channels with 20 μm heightat a flow rate of 8 μl/min followed by thoroughly rinsing with DI water.As the inter-wire spacing of the pSiNWs was approximately 250 nm, it wasexpected that 75 nm nanobeads would be isolated and trapped, while 400nm nanobeads would be size excluded. After isolation, the greenfluorescence from the channel with 20 μm height flowing 75 nm nanobeadswas much stronger than that of the original beads flow and pSiNWsbackground (FIG. 10E), indicating that 75 nm nanobeads were efficientlycaptured and trapped inside the pSiNWs. On the contrary, the bluefluorescence from 400 nm nanobeads was extremely weak and very close tothe pSiNWs background (FIG. 10F), demonstrating that the pSiNWs forestscould barely capture 400 nm nanobeads. The SEM image further validatedthat many 75 nm nanobeads and very few 400 nm nanobeads were capturedand tapped inside the pSiNWs forests (FIG. 10G, in circles). The aboveexperimental results prove that the pSiNWs forest with ˜250 nminter-wires spacing in curved channels with 20 μm height can effectivelyand specifically isolate 75 nm nanobeads.

In addition to channel height, flow rate can also affect the captureefficiency of viruses. In general, high flow rate is preferred as it cansignificantly shorten sample processing time and increase samplecapacity. However, in the present design, the nanoparticle-pSiNWs forestinteractions might be impaired, which accordingly might decease thecapture efficiency of nanobeads. On the contrary, strong local vorticesmight not form at low flow rate, although relatively low flow rate canensure the full contact and interactions between nanoparticle and pSiNWsforest. Therefore, to study the effect of flow rate on the captureefficiency of 75 nm and 400 nm nanobeads, respectively, flow ratesranging from 2 μl/min to 16 μl/min were tested. As shown in FIG. 10H,the capture efficiency of 75 nm nanobeads reaches the maximal value of14.1% at 8 μl/min. When the flow rate is slower than 8 μl/min, captureefficiency of 75 nm nanobeads can be gradually improved by increasingflow rate. At relatively low flow rate, local vortices of fluid flowcould be strengthened by increased flow rate, and thus could effectivelybring nanobeads into pSiNWs forest. However, once the flow rate reachesor surpasses 12 μl/min the capture efficiency of 75 nm nanobeadsdecreases significantly. As a result, at relatively high flow rates,nanobeads might not have sufficient time to pass through inter-wirespacing to be trapped before coming out from the device outlet. In thecontrol group using 400 nm nanobeads, which barely got trapped insidethe pSiNWs forest, the capture efficiency slightly fluctuates between 2%and 4.5% (FIG. 10H).

In a cyclic iteration application, a sample might be run for multipletimes over the device to increase the capture efficiency (Wan, Y. etal., Cancer Research 2010, 70(22):9371-9380). This strategy has beenwidely used to isolate rare molecules. In a previous study, it was foundthat after a single run, green fluorescence from 75 nm nanobeads in thecurved channels displayed random non-uniform distribution (FIG. 10C),indicating there were still considerable empty spacing inside the pSiNWsforest for trapping 75 nm nanobeads. Hence, it was speculated that thecapture efficiency of 75 nanobeads could be significantly improved byincreasing cycle times. In the following experiment, the nanobeadssuspension was recollected from the device outlet and reinjected intothe device for up to 5 times. The capture efficiency of 75 nm nanobeadssignificantly increased from 14.1% to 37.6% after the fifth recycling.In contrast, the capture efficiency of 400 nm nanobeads was onlyslightly increased from 4.5% to 12.9% (FIG. 10I).

Compared to solid silicon nanowire, pSiNWs have been demonstrated to bebiodegradable in alkaline solution including PBS due to its mesopores atnanoscale (Equation 6) (Anderson, S. H. C. et al., physica status solidi(a) 2003, 197(2):331-335). However, they cannot be degraded in DI water(Anderson, S. H. C. et al., physica status solidi (a) 2003,197(2):331-335). The degradation of pSiNWs will allow captured andtrapped nanobeads to escape from the forest. 1×PBS continuously flowsthrough the pSiNWs embedded microfluidic device for 24, 48 and 72 hoursat RT, respectively, and the appearance of pSiNWs forest at eachtimepoint are shown in FIG. 11A and FIG. 11B. SEM images reveal that thedegree of corrosion is dependent on time, and pSiNWs forest totallydegraded at the 72 hour timepoint. The feasibility of releasing captured75 nm nanobeads was tested. After continuous flushing within 1×PBS for24 hours, green fluorescence are barely detectable (FIG. 11C),indicating trapped 75 nm nanobeads escaped from the forest and flowedout of the channels. The extended flushing did not significantly improvethe releasing efficiency, and thus 24 hours was chosen for the followingexperiments. While flushing within DI water in the control group, greenfluorescence remained similar to that of 0 hour (FIG. 12C), indicatingnanobeads trapped in the pSiNWs forest will not be flushed out by flowonly. It was noted that after release of captured 75 nm nanobeads with1×PBS, the recovery efficiency of nanobeads was not investigated astheir concentrations in PBS was too low to be detected by a microplatereader.Si+2OH⁻+4H₂O→Si(OH)₂ ²⁺+2H₂+4OH  (Eqn. 6)Capture and Release of Influenza Virus Using pSiNWs Forest EmbeddedMicrofluidic Device

After optimizing operation parameters using nanobeads, the pSiNWs forestembedded microfluidic device was used to capture H5N2 avian influenzaviruses, and an in situ immunofluorescence assay was used to detect thecaptured viruses (FIG. 12A through FIG. 12C). The time-scale of pSiNWsdegradation in 1×PBS solution was hours, while the whole capture processtakes only 30 min. Therefore it is safe to suspend viruses into 1×PBSfor capturing (Wang, Z. et al., Lab on a Chip 2013, 13(15):2879-2882;Chiappini, C. et al., Advanced Functional Materials 2010,20(14):2231-2239). For the experimental group, 40 μl of H5N2 virussuspension was injected into the device under various flow rates rangingfrom 2 μl/min to 16 μl/min with 3 cycles. Similar to the captureefficiency of 75 nm nanobeads as they have very close sizedistributions, the highest capture efficiency of H5N2 was also achievedat 8 μl/min (FIG. 12D). The fluorescence intensity was approximately 2.5times higher than that in the negative control group which contained noviruses (FIG. 12A and FIG. 12B). SEM images also demonstrated H5N2viruses were captured and trapped inside the pSiNWs forest (FIG. 12C).To further quantitatively determine the capture efficiency of viruses,respective CT values of each group were measured on the basis of RT-qPCRresults, and the capture efficiency of viruses was determined using thedevice was 48±4% (FIG. 12E, FIG. 13).

FIG. 13, comprising FIG. 13A and FIG. 13B, depicts the results ofexperiments demonstrating virus capture and analysis by carbonnanotube-size tunable enrichment platform (CNT-STEP). (FIG. 13A)Schematic illustration showing the design and operation of CNT-STEP. Aswab sample containing viruses (red spheres) is collected from a bird(1). As the virus suspension generated from the swab sample flowsthrough CNT-STEP, the viruses are captured within the sidewall of thedroplet-shaped microfluidic chamber made of CNT forest (2). The CNTforest traps virions with size similar to CNT gap size. After capture,the virus is enriched inside CNT forest and can be characterized byvarious methods (3). (FIG. 13B) Images with increasing magnifications ofCNT-STEP before PDMS bonding. Brightfield microscopy image showing topview of the droplet-shaped CNT microfluidic chamber on silicon substrate(1). SEM image of CNT porous microfluidic sidewall in a tilted view (2).High magnification SEM image showing side view of CNT forest structure(3). TEM image of a single aerosol-assisted chemical vapor deposition(AACVD) synthesized CNT (4). Scale bars in (B) 1-4 are 2 mm, 50 μm, 50nm, and 10 nm, respectively.

FIG. 14, comprising FIG. 14A through FIG. 14D, depicts the results ofexperiments demonstration the characterization of the growth, geometry,and size-tunable filtration properties of the CNT-STEP. (FIG. 14A)Diameter and (FIG. 14B) density of the iron nanoparticles (red) and CNT(black) as functions of the thickness of the iron catalyst thin films (1nm, 3 nm, 6.5 nm, 9.5 nm, and 12 nm, n=8 each). Iron nanoparticles wereformed during temperature ramping phase (from room temperature to 825°C. in 30 minutes duration) before the CNT synthesis. The diameter anddensity were measured from SEM images. (FIG. 14C) Calculated the gapsize of the CNT structures from the measured densities and diameters.The inset illustrates the square geometry model used for thecalculation, where CNT-to-CNT distance (G) and diameter of CNT (D) areconstants. (FIG. 14D) Measured size-tunable filtration characteristicsof CNT-STEP with various gap sizes of 25 nm, 85 nm, and 280 nm. Smallmolecule fluorescein and fluorescent polystyrene nanospheres of 20 nm,50 nm, 100 nm, 140 nm, 400 nm, and 1000 nm in diameter were used.Penetration ratio is defined as the ratio of the fluorescence intensityof the filtrate to that of the original suspension (n=8).

FIG. 15, comprising FIG. 15A through FIG. 15G, depicts the results ofexperiments demonstrating H5N2 avian influenza virus (AIV) captured anddetected by the CNT-STEP microdevice. (FIG. 15A) Fluorescence microscopeimages of on-chip IFA of H5N2 AIV inside the CNT-STEP microdevices with25 nm, 85 nm, and 280 nm gap sizes. Red arrows indicate the flowdirection. (FIG. 15B) RT-qPCR curves of H5N2 (10⁵ ELD₅₀ titration)trapped and extracted from 25 nm, 85 nm, and 280 nm gap size deviceafter filtration. (FIG. 15C) SEM images of the H5N2 AIV virions trappedinside the CNT forest structure of 25 nm gap size (the flow direction isinto the plane), with inset at a higher magnification (scale bars: 200nm). (FIG. 15D) TEM images of H5N2 AIV around MWCNTs after the CNTstructures were scraped from the microdevice, with inset showing theindividual virion at a higher magnification (scale bars: 200 nm). (FIG.15E) RT-qPCR plots of H5N2 AIV samples of 0.1 ELD₅₀ titration afterenrichment and 1 and 10 ELD₅₀ titrations before enrichment (n=6). (FIG.15F) Pie charts presenting the percentage distribution of NGS readsgenerated by blast search before and after microdevice enrichment. (FIG.15G) Circos plot of assembled H5N2 contigs generated by NGS from theCNT-STEP microdevice enriched sample. Track 1 (outermost): Referencestrain “A/mallard/Wisconsin/411/1981(H5N2)” (the closest strain of H5N2by blast search). Track 2: Variants with color coding (deletion: longblack line; transitions: A-G: fluorescent green, G-A: dark green, C-T:dark red, T-C: light red; transversion: A-C: brown, C-A: purple, A-T:dark blue, T-A: fluorescent blue, G-T: dark orange, T-G: violet, C-G:yellow, G-C: light violet). Track 3: Open reading frame (green). Track4: Location of AIV detection by Sanger's method targeting the NP gene(713˜1463 bp). Track 5: Coverage depth at each genomic position (black)(scale of the plot from 0 to 30k reads). Track 6: Contigs generated bydeveloped pipeline de-novo assembly (grey). Inset is a TEM picture ofthe virus.

FIG. 16 depicts the setup of aerosol assisted chemical vapor deposition(AACVD) for CNT synthesis. Top image: Image of AACVD setup inside a fumehood. Bottom image: Schematic illustration of AACVD for the CNTsynthesis.

Afterwards, after 24 hours continuously flushing with 1×PBS, the greenfluorescence intensity was only ˜10% of that before flushing. In thecontrol group, DI water which cannot dissolve pSiNWs was used, and thegreen fluorescence intensity only decreased to ˜80% after 24 hoursflushing (FIG. 14A). This result indicated that captured viruses werereleased by degradation of pSiNWs forest and then flushed out in PBSflow. In contrast, simply flushing using DI water which hasinsignificant effect on degradation of pSiNWs is inefficient to directlyremove trapped viruses from pSiNWs forest. The released viruses in PBSwere recollected for further identification and culture. SandwichELISA-like virus identification was performed first. Released viruseswere enriched with H5 antibody-conjugated magnetic beads (FIG. 15Athrough FIG. 15C) and stained with red fluorescence labeled H5antibodies for viral identification. Red fluorescence intensity inexperimental group was ˜2 times higher than that of negative controlgroup containing pSiNWs degradation solution only without any viruses,indicating released viruses can be successfully recollected foridentification. To determine the recovery efficiency, CT values ofRT-qPCR results were used to quantify viral concentration in thereleasing solution (Equation 5, FIG. 14B, FIG. 16). It was found therecovery efficiency was 29±7% in releasing solutions with varying volumeranging from 60 μl to 1000 μl. So about 60% of captured viruses could berecovered and recollected. In recovery sample A, the CT value was16.8±0.6. After conversion, virus titer was ˜6×10⁶ EID₅₀/ml. Such highconcentration would satisfy the following virus cultivation if virusesare alive. To test the viability, released viruses were inoculated intoembryonated chicken eggs for cultivation, and HA test was used toquantify the relative concentration of harvested virus solution. Afterbeing diluted 2¹⁰ times, virus suspension failed to agglutinate the redblood cells, and RBCs began to settle out of suspension forming redprecipitation. Thus the titer of the virus sample was determined to be1:2⁹, which is ˜4×10⁶ EID₅₀/ml (The CT value of virus sample was 1:2¹⁰was 16.45) (FIG. 14C). The findings demonstrated that by propagatingthese viable viruses after capture and release cycles, large amounts ofviruses could be obtained for various subsequent virus-related studies.

In conclusion, a pSiNWs forest embedded microfluidic POC device wassuccessfully developed for label-free capture and release of viruses.For the first time it was demonstrated that targets at nanoscale can bephysically trapped into inter-wire spacing with tunable distance withDean flow in curved channels. Approximately 50% of viruses can bephysically captured and trapped in pSiNWs forest after only 3 iterativecycles at as high as 8 μl/min flow rate. Viruses remain viable after 24hours and can be released through the degradation of the pSiNWs forest.The recovery efficiency is approaching 29% of total inducing virus.Moreover, it was also demonstrated that released viruses can be lysedfor RT-qPCR or further cultivated with embryonated chicken eggs. Withthis POC device, viruses with specific sizes could be isolated in 30minutes and recovered by dissolving pSiNWs in PBS for another 24 hours.Recovered viruses are alive and can be further cultivated withembryonated chicken eggs for further long term analysis, like genemutation detection, drug testing.

Example 2: A VACNT Integrated Handheld Device for Label-Free VirusCapture, Detection, and Enrichment for Genomic Analysis

A handheld (1 cm×2 cm) vertically aligned carbon nanotube (VACNT)integrated microfluidic device is presented herein to capture virus bynanoscale filtration. The device contains porous herringbone array madeof VACNT with a tunable gap size (20-550 nm) on a fused silicasubstrate. Avian influenza virus (AIV) H5N2 subtype was isolated fromchicken swab samples and detected by on-chip immunofluorescencestaining. The device enriched the H5N2 from swab samples and improvedthe RT-qPCR detection limit by at least one order of magnitude,confirmed by SEM and gel electrophoresis. Finally, the isolated H5N2 wassuccessfully cultured ex vivo inside chicken eggs.

FIG. 17 depicts the results of experiments demonstrating the temperatureprofile of the furnaces during the CNT synthesis. The devices wereloaded at the center region (˜3 inches in length) of the 2^(nd)furnaces.

VACNT was synthesized by aerosol-based chemical vapor deposition (CVD)on a lithography-patterned iron-catalyst thin film to form a porousherringbone array with 50 μm in height (FIG. 17A) (Stroock et al.,Science, 2002, 295:647-651). The porous structure captured virus withdiameter similar to VACNT gap size during the enhanced mixing process.After virus was captured, immunofluorescence or RNA extraction afteron-chip virus lysis could be performed on-chip. Furthermore, theenriched H5N2 was collected by scratching the VACNT forest structurewith a pipette tip and directly inoculated into chicken eggs for ex vivoculture. Optical, SEM, and TEM images demonstrated the assembled devicewith vertically aligned CNT structure selectively grew on fused silicasubstrate at different scales (FIG. 17B).

FIG. 18, comprising FIG. 18A through FIG. 18E, depicts the results ofexperiments demonstrating the effect of the synthesis time on theheight, CNT diameter, and density of the vertically aligned CNTstructure. Geometrical parameters were measured from SEM images. (FIG.18A) Height of VACNT structure synthesized for 30 minutes on 1 nm, 3 nm,6.5 nm, 9 nm, and 12 nm thick iron thin films (n=8). (FIG. 18B throughFIG. 18D) The effect of the synthesis time on (FIG. 18B) height, (FIG.18C) CNT diameter, (FIG. 18D) CNT density of the VACNT structure. TheCNT was grown on 3 nm, 6.5 nm, and 12 nm thick iron catalyst thin filmsunder 5, 10, 20, 30, and 40 minutes of AACVD synthesis (n=8). (FIG. 18E)Histograms of the CNT diameter formed on 1 nm, 3 nm, 6.5 nm, 9 nm, and12 nm thick iron thin films. The analysis was based on SEM images. Thehistograms were fitted for normal distribution in solid lines. The insetis a TEM image of AACVD synthesized CNT on 12 nm thick iron thin film.

FIG. 19, comprising FIG. 19A and FIG. 19B, depicts the results ofexperiments demonstrating the characterization of the filtration relatedproperties of the vertically aligned CNT structure. (FIG. 19A) Porositycalculated from cylindrical pillar arrangement model and SEM images ofCNT growing on 1 nm, 3 nm, 6.5 nm, 9 nm, and 12 nm thick iron catalystthin films for 30 minutes AACVD synthesis (n=8). The inset illustratesthe geometry model of the CNT forest. (FIG. 19B) Calculated porosity ofCNT grew on 3 nm, 6.5 nm, and 12 nm thick iron catalyst thin films,based on geometry assumption plotted in inset of (FIG. 19A).

FIG. 20 depicts the testing setup of the CNT-STEP microdevice. The virussample was loaded into the inlet sample reservoir and processed throughCNT-STEP microdevice via a vacuum source connected through a waste trapat the outlet. The vacuum pressure was measured by a miniature pressuresensor and regulated by a precision mechanical regulator.

Cross-sectional SEM images (FIG. 18A) were analyzed and it was observedthat the nanoscale geometry of VACNT could be controlled by ironcatalyst thickness. CNT diameter and density can be turned in the rangeof 14˜45 nm and 2×10⁸-5×10¹⁰/cm², respectively (FIG. 18B and FIG. 18C).By using diameter and density data, it was calculated that the gap sizeof the VACNT forest has a range of 20-550 nm (FIG. 19A). To study thesize-based capturing process, a mixture of fluorescent polystyrenenanospheres of 20 nm (red), 100 nm (green), and 1000 nm (blue) indiameter were added into 95 nm gap devices and the flow-through wascollected under a flow rate ranging from 0.1˜100 μl/min. In general, 100nm and 20 nm nanospheres could be captured and the capture efficiencyincreased with flow rate. 100 nm nanospheres had higher captureefficiency (˜16%) at 100 μl/min flow rate (FIG. 19B and FIG. 19C).

Clinically relevant samples were constructed by spiking H5N2 virus (FIG.20A) into a swab medium collected from a healthy (virus-free) chicken.200 μl of H5N2 swab sample were added into a 95 nm device at a flow rateof 100 μl/min and on-chip immunofluorescence assay was applied. Stronggreen fluorescent signals indicated that H5N2 was trapped inside theherringbone structure (FIG. 20B), which was later confirmed by SEMimages (FIG. 20C). Virus was lysed on-chip and RNA extracted for geneticdetection. FIG. 20D shows that a signature DNA fragment (750 bp) wasobserved in the all H5N2 samples and the positive control, but not thenegative control.

FIG. 21 depicts SEM images of cross-sectional views of CNT structuregrowing on 1, 3, 6.5, 9, and 12 nm thick iron catalyst layer. SEMmagnification: 2,000 (top) and 20,000 (bottom). (Scale bars: 500 nm(top), 100 nm (bottom)).

More importantly, the device demonstrated an order of magnitudeimprovement in the detection limit of the current golden standard ofvirus detection, the RT-qPCR. 1 mL of 0.1ELD₅₀ H5N2 sample was addedinto the 95 nm gap device, RNA was extracted, and RT-qPCR was run forRNA detection (FIG. 21, red lines). By comparing with 0.1ELD₅₀ (bluelines) and virus sample 1ELD₅₀ (black lines) virus samples, thedetection limit of RT-qPCR was improved by at least one order ofmagnitude. Finally, after enrichment by the device, the scratched VACNTstructures with virus was inoculated into chicken eggs, and H5N2 wassuccessfully propagated after three days of ex vivo culture.

Example 3: Tunable and Ultra-Sensitive Virus Detection Using CarbonNanotube Arrays

Viruses may cause unpredictable and recurring outbreaks that lead todevastating mortality and traumatic economic losses, as exemplified bythe 1918 influenza pandemic, the ongoing battle against HIV/AIDS, andthe most recent Ebola and Zika outbreaks (Fauci, A. S. et al., New Engl.J. Med. 2012, 366:454-461). However, there is still a large pool ofunknown mammalian and human viruses, among which could be critical viralpathogens (Anthony, S. J. et al., mBio 2013, 4; Woolhouse, M. et al.,Philos. T. Roy. Soc. B 2012, 367:2864-2871). Almost all lethal viraloutbreaks in the past two decades were caused by new emerging viruses(Chiu, C. Y. et al., Curr Opin Microbiol 2013, 16:468-478). As over 50%of the human pathogens are known to be zoonotic (Howard, C. R. et al.,Emerg Microbes Infect 2012, 1:e46; Mark, E. J. W. et al., Emerg. Infect.Dis. 2005, 11:1842), virus samples can be originated from varioussources, e.g. human, animals, and different environments. Thus, it isclear that the successful virus isolation, identification, and genomecharacterization, directly from field and clinical samples will lead torapid discovery of emerging viral pathogens (Pennington, H., Nat. Rev.Micro. 2004, 2:259-262).

Since the high mutation rate and the genetic diversity of viruseswarrant extensive surveillance (King, D. A. et al., Science 2006,313:1392-1393), various virus detection approaches have beenestablished: i) enzyme-linked immunosorbent assay (ELISA) (Yolken, R.H., Yale J. Biol. Med. 1980, 53:85-92), ii) polymerase chain reaction(PCR) (Ellis, J. S. et al., Rev. Med. Virol. 2002, 12:375-389; Spackman,E. et al., J. Clin. Microbiol. 2002, 40:3256-3260), iii) virus isolation(Eisfeld, A. J. et al., Nat. Protocols 2014, 9:2663-2681; Wood, J. M. etal., Nat. Rev. Micro. 2004, 2:842-847), and iv) next generationsequencing (NGS) (Chiu, C. Y. et al., Curr Opin Microbiol 2013,16:468-478; Radford, A. D. et al., J. Gen. Virol. 2012, 93:1853-1868).However, additional advancements in the sample preparation techniques toenrich and concentrate viruses are urgently needed (Beerenwinkel, N. etal., Front. Microbiol. 2012, 3:16; Chin, C. D. et al., Lab Chip 2012,12:2118-2134; Heider, S. et al., Virology 2014, 462-463:199-206; Li, L.et al., J. Virol. Methods 2015, 213:139-146; Noda, T., Front Microbiol2011, 2:269). In addition, the most conventional virus-samplepreparation protocols utilize immunological capture, physical separationor a combination of both (van Reis, R. et al., L. Membrane Sci. 2007,297:16-50; Yeh, Y.-T. et al., Ann Biomed Eng, 2014, 1-11).Unfortunately, immunological capture requires prior knowledge of thetargets, thus it is not appropriate for virus discovery and can lead totechnical difficulties for identifying new or emerging virus strains.Ultracentrifugation is the most commonly used physical method for virusenrichment and concentration. Unfortunately, it involves bulkyequipment, intensive labor, lengthy sample preparation, and haslimitations for concentrating small amounts of viruses in minute volumes(Radford, A. D. et al., J. Gen. Virol. 2012, 93:1853-1868; Yeh, Y.-T. etal., Ann Biomed Eng, 2014, 1-11; Bibby, K., Trends Biotechnol. 2013,31:275-279). Microfiltration membranes can remove large particles withinsamples while keeping the virus particles in the supernatant. It isnormally used as one of the steps in the whole sample preparationprotocol for virus analysis, however it neither removes contaminants ofsmall size (e.g. nucleic acids and proteins) nor concentrates the sample(Daly, G. M. et al., PLoS ONE 2011, 6:e28879; Hall, R. J. et al., J.Virol. Methods 2014, 195:194-204; Rosseel, T. et al., J. Virol. Methods2015, 222:72-80). Although ultrafiltration membranes are widely used asan essential viral clearance step in the biopharmaceutical productionfrom human or animal origin (van Reis, R. et al., L. Membrane Sci. 2007,297:16-50; DiLeo, A. J. et al., Nat. Biotech. 1992, 10:182-188), theirusage for virus detection is rare primarily due to their low porosity,high operation pressure, poor virus viability and difficulty in virusaccess for further analysis.

In this context, robust arrays of aligned CNTs with controlledinter-tube distance could be used to effectively trap/concentrateviruses within a 3-dimensional porous system. Although CNTs have beenused as biochemical sensors (Balasubramanian, K. et al., Anal. Bioanal.Chem. 2006, 385:452-468), imaging probes (Liu, Z. et al., Nano Res.2009, 2:85-120), drug delivery vehicles (Cai, D. et al., Nat. Methods2005, 2:449-454), X-ray sources (de Heer, W. A. et al., Science 1995,270:1179-1180), neuron protection (Lee, H. J. et al., Nat. Nano. 2011,6:121-125), treatment of drug addiction (Xue, X. et al., Nat. Nano.2016, 11:613-620), and substrates for immunological capture of mammaliancells and bacteria (Chen, G. D. et al., Small 2011, 7:1061-1067), theyhave not been integrated into tunable devices able to isolate viruses ofdifferent sizes. Here, a reliable, scalable CNT-STEM technology isdeveloped that provides size-based, label-free, viable enrichment andconcentration of viruses from field samples. The following studysynergistically integrates bottom-up controlled nanotube synthesis withtop-down microfabrication. The study demonstrates that the CNT-STEM notonly improves virus detection sensitivity and the minimal virusconcentration for virus isolation by at least 100 times (FIG. 22A andFIG. 22B), but also removes host and environmental contaminants, andconcentrates samples to enable direct virus identification by NGS fromfield-collected samples.

The materials and methods are now described.

Patterning of Iron Catalyst Thin Film

As shown in FIG. 23A, a 4″ single-side polished prime silicon wafer wascleaned in piranha solution, acetone, isopropyl alcohol (IPA) andultrapure water sequentially. Lift-off photoresist LORSA (MicroChem) andnegative photoresist NFR105G (JSR Microelectronics) were spin-coated at4000 rpm, respectively, followed by photolithography patterning with acontact aligner (Karl Suss MA/BA6). Iron was deposited by an e-beamevaporator (Semicore) under 10⁻⁷ mTorr vacuum with deposition rate of0.1 nm per second to target thickness of 1, 3, 5, 8, 10 nm. The actualthickness of the deposited films was measured to be 1.0±0.1, 3.0±0.2,6.5±0.5, 9.2±0.4, 11.9±0.8 nm by AFM (Bruker Dimension Icon). A thinlayer of negative photoresist NFR105G was spin-coated as a protectivelayer prior to dicing. The silicon substrate was then diced intoindividual dies of 1.2 cm by 1.2 cm by a dicing saw (Advanced DicingTechnologies). Photoresist was lift-off by soaking the substrate insideremover-PG (MicroChem) overnight at 60° C.

N-MWCNT Synthesis by AACVD

The aerosol-assisted chemical vapor deposition (AACVD) setup consistedof an ultrasonic nebulizer (RBI instrumentation, Meylan, France), twotube furnaces (Thermo Scientific) arranged in series and a waste trapfilled with acetone (FIG. 24). Benzylamine (Fluka, CAS:100-46-9) was fedthrough the system by a nebulizer, working as both carbon source andnitrogen dopant. The iron-patterned substrates were placed in the secondfurnace. All components were airtight sealed by silicone paste (McMasterCarr) and flushed with argon and 15% hydrogen of 0.5 L/min flow rate for5 minutes. The furnace temperature ramped to 825° C. in 30 minutes. Whenthe temperature reached 825° C., the nebulizer was turned on and theargon and 15% hydrogen flow was increased to 2.5 L/min. After thesynthesis process was completed, the nebulizer was turned off, the flowrate of carrier gas was decreased back to 0.5 L/min and the furnacetemperature was set back to 25° C. The cooling process usually took 3hours for the furnaces to reach room temperature.

CNT-STEM Assembly and Experimental Setup

The N-MWCNT forest pattern was designed as a droplet-shape to distributethe aqueous sample evenly. The patterned N-MWCNT structure on thesilicon substrate was integrated into a microfluidic device by bondingit with a PDMS chamber. The PDMS chamber was fabricated by standard softlithography (Xia, Y. et al., Annu. Rev. Mater. Sci. 1998, 28:153-184).The mold was fabricated by patterning SU-8 on silicon wafers with acontact aligner (Karl Suss MA/BA6). The ratio of the depth of the PDMSchamber and the height of the N-MWCNT forest was ˜0.8-0.9. Then,well-mixed PDMS precursor (part A: part B=10:1, Sylgard 184, DowCorning) was poured onto the SU-8 mold and bake at 60° C. for 40minutes. The partially cured PDMS layer was diced into 1.2 cm by 1.2 cmsquares with a razor blade. Two through-holes serving as inlet andoutlet, 4 mm and 1 mm in diameter respectively, were punctured throughthe PDMS using a Luer adapter (BD). Before bonding, both the PDMSchamber and the N-MWCNT patterned silicon substrate were treated with RFoxygen plasma (M4L, PVA TePla Inc) with processing parameters of 400mTorr oxygen pressure, 50 W power, and 30 seconds duration. Then theywere aligned and gently pressed together and baked at 85° C. for fourhours.

The experimental system for the CNT-STEM includes a sample reservoir, awaste trap, and components for flow actuation and control (FIG. 23B). A100 cylindrical sample reservoir formed with silicone tube of 5 mm innerdiameter was attached to the inlet port. The outlet port was connectedto a custom-made waste trap using a silicone tube of 0.5 mm innerdiameter. The waste trap had three connections to a vacuum pump, aminiaturized pressure sensor, and a mechanical pressure regulatorrespectively. The vacuum pump (McMaster-Carr) provided a negativepressure. The miniaturized pressure sensor (Honeywell ASDXL) had asensing range of 10 inches of water column. It was soldered to a printedcircuit board (McMaster-Carr) and powered by a 9V battery. Themechanical pressure regulator (McMaster-Carr) regulated the vacuumsuction. Fluidic connections were sealed by applying uncured PDMSprecursor as sealant to the ends of the connections, and then curedunder room temperature for 24 hours.

Characterization of N-MWCNT Forest Geometries of Vertically AlignedN-MWCNT by AACVD Synthesis

The nanoscale geometries of the N-MWCNT synthesized on 3 nm, 6.5 nm and12 nm thick iron catalyst thin films under different synthesis time of5, 10, 20, 30 and 40 minutes were studied by cross-sectional SEM imagesof the N-MWCNT structure taken by a field emission SEM with acceleratingvoltage of 5 kV (LEO 1530 FESEM). The height of the N-MWCNT forest wascharacterized with cross-sectional SEM images under 8×10² magnification.For diameter analysis of single N-MWCNT, 6×10⁴ magnification was usedand a total number of 500 focused N-MWCNTs were measured with ImageJ.Both the N-MWCNT diameter D and its probability density function ƒ(D)were calculated. For density and inter-tubular distance measurement ofthe aligned N-MWCNT structure, the images were taken under 2.5×10⁴magnification at bottom of the N-MWCNT forest close to the substrate.One line equivalent to 1 μm in length was drawn perpendicular to theN-MWCNT growth direction on each image. The numbers of focused N-MWCNTsthat crossed the line was counted to calculate its linear density λ. 20images of each synthesis condition were analyzed and five 1 μm lineswere drawn for each image. For the inter-tubular distance, the distancewas measured between pairs of neighboring focused N-MWCNTs that werecrossed by the drawing line. 20 images of each synthesis condition werecollected and data on 5 drawing lines on each image were analyzed foreach image. Assuming the N-MWCNT density is isotropic in 2D, theporosity Φ can be calculated from the measured N-MWCNT line density Δ,diameter D_(i) and the probability density function of the diameterƒ(D_(i)):

$1 - {\frac{\pi}{4}\lambda^{2}{\sum\limits_{i}{{f\left( D_{i} \right)}{D_{i}^{2}.}}}}$Measurement and Analysis of the Iron Nanoparticle Geometry

To study the geometrical properties of the iron nanoparticles and therelationship to those of the N-MWCNTs, silicon wafers were diced intodevice dies and iron catalyst thin films of targeted thicknesses of 1,3, 5, 8, 10 nm were deposited on different device dies. For one set ofdevice dies with different thicknesses of the iron catalyst film, theAACVD process went through the thermal ramping stage and was terminatedprior to feeding the precursor (benzylamine). The SEM images of the topview of the iron nanoparticles were taken under 5×10⁴ magnification. SEMimages of iron particles were analyzed for their size and spatialdistribution using Matlab image processing toolbox. The averageparticle-to-particle distance was calculated by applying the Delaunaytriangle selection to determine the closest neighbor particles, thenrepresented as the mean of the three edges r₁, r₂ and r₃ (FIG. 25)(Bray, D. J. et al., J. R. Stat. Soc. Ser. C-Appl. Stat. 2012,61:253-275).

Raman Spectra Measurement of N-MWCNT

AACVD synthesized N-MWCNT was characterized by Raman microscopy(Renishaw, InVia Raman microscopy) using 514 nm laser excitation for 30seconds under 50× magnification. The laser power to the sample was 10μW.

CNT-STEM Filtration Process

The assembled CNT-STEM was primed by adding 10 μL of 0.5% Tween-20(Sigma-Aldrich) at the inlet port and letting the device sit undisturbeduntil all the air inside the device was replaced by Tween-20. Thiswetting process took around 15 minutes. Subsequently, another 50 μL of0.5% Tween-20 was added to inlet port. The vacuum suction from theoutlet was turned on and the differential pressure was maintained at 0.1psi (6.9×10² Pa) to move the aqueous phase through the CNT-STEM. In themeantime, device leakage was tested by estimating the travel speed ofthe air-liquid interface inside the silicone tubing. If the devicepassed the leak test, 200 Dulbecco's phosphate buffered saline (DPBS)(Cellgro) was added to wash the device. After most of the DPBS flewthrough the device, the virus sample was then added to the inlet portwhile the vacuum suction remained. After most of the virus sample wasfiltered through, 50 μL DPBS were added to rinse the device. All samplescontaining viruses were filtered through member filters (VWR) of 0.2 μmor 0.45 μm pore size for swab samples and tissue samples beforeintroducing the filtrates using the CNT-STEMs.

N-MWCNT Inter-Tubular Distance Characterized by Nanoparticle Penetration

Fluorescein solution (Sigma-Aldrich, #46955) and polystyrene nanospheresuspensions (Thermo Scientific Inc.) were diluted by 0.5% Tween-20 intofinal concentration of 0.01% (solid). After device priming, 20 μL of thesuspension were loaded at the inlet port. The vacuum suction was turnedoff after all the suspension was transported into the device. Thefluorescence image of the device was taken by an sCMOS camera (HamamtsuORCA-Flash4.0 V2) connected to a fluorescence microscope (Olympus IX71).The fluorescence intensity was calibrated and measured by ImageJ(Bankhead, P. Analyzing fluorescence microscopy images with ImageJ.(2014)). The penetration ratio was defined by the ratio of fluorescenceintensity outside the CNT droplet-shaped chamber (I_(in)) to that inside(I_(out)), both corrected with background fluorescence intensity(I_(bg)) without the fluorescent agents:

${{Pennetration}\mspace{14mu}{Ratio}} = {\frac{I_{out} - I_{bg}}{I_{in} - I_{bg}}.}$Size Measurement of Nanospheres and AVI Virions

The diameters of fluorescent polystyrene nanospheres were measured by aNano ZS particle-size analyzer (Malvern Zetasizer, Malvern InstrumentsLtd, UK). The size distribution of the nanospheres was calculated by theaccompanying software (Nanov510) using a refractive index of 1.59.

Similarly, 10⁷ EID₅₀/mL AIV solution was diluted by 1000 fold with 20 mMphosphate buffer at pH 7.4. The suspension was then passed throughmembrane filters of 0.45 μm (Celltreat scientific products) and 0.2 μm(VWR) pore size sequentially, then analyzed on a Nano ZS particle-sizeanalyzer (Malvern Zetasizer, Malvern Instruments Ltd, UK). By assumingrefractive index of 1.48 (Wang, S. P. et al., Proc. Natl. Acad. Sci.U.S.A 2010, 107:16028-16032), the Nanov510 software converted intensitydata into diameter measurements.

H5N2 AIV Propagation and Sample Preparation

H5N2 AIV was propagated in specific pathogen-free (SPF) embryonatedchicken eggs (ECE) via allantoic cavity route inoculation at 9-11 daysof age. The inoculated eggs were placed in a 37° C. egg incubator for 72hours. Then the eggs were removed from the incubator and chilled at 4°C. for 4 hr. Each egg was cracked open at the top air sac. The shellpeeled without breaking the air sac membrane. The allantoic fluidcontaining the virus was harvested using a 3 mL sterile syringe with a25G×⅝″ needle. The harvested allantoic fluid was clarified bycentrifugation at 8000 rpm for 5 minutes. The virus titers were measuredin embryo infectious doses 50% (EID₅₀) by the Reed-Muench method (Reed,L. J. et al., Am. J. Epidemiol. 1938, 27:493-497). Briefly, the freshlypropagated H5N2 AIV stock was prepared in 10-fold serial dilutions from10¹ through 10⁹. Each dilution was inoculated into 5 eggs, 0.1 mL peregg. The inoculated eggs were incubated at 37° C. for 72 hours. The eggswere candled daily to remove dead eggs to chill them at 4° C.refrigerator. After 72 hours of incubation, allantoic fluid washarvested from each egg and spin down. The supernatant was collected andpassed through a membrane filter of 0.2 μm pore size prior to use. Theinfection status of each egg was determined by Dot-ELISA. AIV H5N2samples were produced experimentally by spiking a freshly propagatedLPAIV H5N2 strain (A/chicken/PA/7659/1985) into tracheal swabs obtainedfrom SPF chickens.

On-Chip Immunofluorescence Assay for H5N2 AIV Detection and FluorescenceIntensity Measurement

After virus capture and phosphate buffered saline (PBS) washing insidethe CNT-STEM, monoclonal antibody of the H5 HA protein (100 μL of 1:1000diluted work solution, Penn State Animal Diagnostic Laboratory) wasadded through the inlet, incubated at 37° C. for 40 minutes, and washedwith 1 mL of PBS. Then goat anti-mouse immunoglobulin conjugated withFITC (100 μL of 1:500 work dilution, KPL) was added and incubated at 37°C. for 40 minutes and followed by 1 mL PBS wash. Fluorescence microscopyimages were obtained by an sCMOS camera (Hamamtsu ORCA-Flash4.0 V2)connected to a fluorescence microscope (Olympus IX71). By measuringaverage intensity of an area of 100 μm×100 μm across the N-MWCNT walls,the fluorescence signal was calculated by ImageJ (Bankhead, P. Analyzingfluorescence microscopy images with ImageJ. (2014)).

Virus Lysis and RNA Extraction

After performing virus filtration by the CNT-STEM, the virus-trappeddevice was disassembled by peeling the PDMS chamber using a razor blade.Normally the N-MWCNT structure remained on the PDMS surface. The N-MWCNTstructure was then scraped from the PDMS chamber with a razor blade andplaced into a microcentrifuge tube containing 50 μL lysis/bindingsolution (MagMax, Life Technologies). The viral RNA was extracted withMagMax™-96 AI/ND viral RNA isolation kit (Life Technologies, Cat. #AM1835) following the manufacturer's protocol.

Real-Time Reverse-Transcriptase Polymerase Chain Reaction (rRT-PCR)

Primers and a probe specific to influenza type A were used (Spackman, E.et al., J. Clin. Microbiol. 2002, 40:3256-3260). The master solution ofrRT-PCR was prepared as a 50 μL reaction mixture using a One Step RT-PCRKit (Cat. No. 210212, QIAGEN, Valencia, Calif.), containing 10 μL oftemplate RNA, 25 μL of RNase-free water, 10 μL of 5×Buffer, 2 μL of dNTPmix (10 mM each dNTP), 1 μL of enzyme mix, and 1 μL of each of the twoprimers. Amplification was performed with a real-time PCR system (7300,Applied Biosystem Inc.) using a reverse transcription step at 50° C. for30 min. The initial PCR activation step was set at 95° C. for 15 min,then followed by 94° C. for 30 s, 50° C. for 30 s, and 72° C. for 90 sof each cycle for 38 cycles, and lastly finished with a single cycle of72° C. for 5 min. The data was collected and processed by themanufacturer's software (7300 V1.4.0, Applied Biosystem Inc.). The Ctvalue was determined by 2^(−ΔΔct) method. The efficiency of the PCR isdetermined by:

${Efficiency}{{= {{10^{\frac{1}{Slope}}} - 1}},}$where Slope is the slope of the PCR standard curve.Virus Isolation and Characterization of the Propagation by DOT-ELISA

N-MWCNTs were collected into a microcentrifuge tube containing 0.1 mL ofPBS and inoculated into an ECE as described for virus propagation. After72 hours of incubation at 37° C., allantoic fluid was harvested. Theallantoic fluid infection status was determined by Dot-ELISA using AIVH5 monoclonal antibody. 10 μL test specimens were applied to a strip ofa nitrocellulose membrane (Thermo Scientific Inc.). After the membranewas air-dried, blocking solution (10 mL of nonfat milk diluted in 30 mLof water) was added and the membrane was incubated at room temperaturefor 10-20 minutes. After blocking, the membrane strip was allowed to airdry. Anti-H5 MAb was then applied and incubated for 60 minutes at roomtemperature, followed by PBS wash for 1-2 minutes with two repeats, andallowed to air dry. Next, goat anti-mouse IgG FITC conjugate was addedat 1:500 working dilution of the 0.1 mg/mL stock solution and incubatedfor 60 minutes at room temperature. The PBS wash step was repeated.Finally, BCIP/NBT (5-bromo-4-chloro-3-indolyl-phosphate/nitrobluetetrazolium, #B8503, Sigma Aldrich) was applied and the membrane waskept in the dark during color development.

Sample Preparation for Next Generation Sequencing (NGS)

To prepare a sample for NGS, first cDNA library was built from the totalRNA extracted of the sample. TruSeq Stranded Total RNA Sample Prep Kit(Illumina, San Diego, Calif., USA. Cat. #RS-122-2201) was used toreverse transcribe the extracted RNA sample (total RNA) into cDNA. Themanufacturer instructions were followed with the exception that theinitial poly A enrichment step was skipped. Briefly, by applyingelevated temperatures, total RNA was fragmented into shorter segments.Those shorter RNA fragments were first reverse transcribed into cDNAstrands with random primers. By adding DNA polymerase I and RNase H, thecomplementary cDNA strands were synthesized. The cDNA was ligated to anadaptor and amplified by PCR to generate cDNA library. The quality ofthe cDNA library was tested by Agilent Bioanalyzer system (AgilentTechnologies, Santa Clara, Calif., USA). Library concentration wasassessed by qPCR using the KAPA Library Quantification Kit IlluminaPlatforms (Kapa Biosystems, Wilmington, Mass., USA). Finally, theprepared cDNA libraries were loaded into different lanes of the MiSeqsequencer using 150 nt single read sequencing (Illumina, San Diego,Calif., USA) to generate raw NGS reads.

De Novo Assembly and Analysis

The overall pipeline for sequence data analysis is summarized in FIG.26. Starting from raw NGS reads, the added sequencing adaptors wereremoved by SortMeRNA. Quality trimming by Trimmomatic (BWA-MEM) was usedto remove matched contaminating sequences of the host (e.g. chicken,turkey, human) and bacteria, as well as rRNA. Unmatched sequence readswere assembled using de novo assembly software SPAdes (V.3.5.0)(Bankevich, A. et al., J. Comp. Biol. 2012, 19:455-477) with Kmer-sizesetting of 85. After de novo assembly, LASTZ (R. S. Harris, Improvedpairwise alignment of genomic DNA. (ProQuest, 2007)) and SAMMtools (Li,H. Bioinformatics 2009, 25:2078-2079) were used to identify and obtainthe final viral consensus sequences. Coverage was analyzed by BWA-MEM.

Phylogenetic Analysis

Phylogenetic tree was generated by MEGA (V.6.06) (Kumar, S. et al.,Computer applications in the biosciences: CABIOS 1994, 10:189-191) usingClustalW alignment and maximum-likelihood (ML) (Tamura, K. et al., Mol.Biol. Evol. 2011, 28:2731-2739). The tree topologies were made bybootstrap analysis with absolute distances following 1,000 bootstrapreplicates (Felsenstein, J. Evolution 1985, 39:783-791).

Intrahost Variant Calling and Analysis

Intrahost variants (iSNVs) were identified using a haplotype-basedvariant detector (Freebayes) with setting of ploidy of 1 and error rateof 0.8% (Illumine MiSeq) (Quail, M. A. et al., BMC genomics 2012,13:341). If the frequency of variant population was higher than 0.8%, itwas considered as an iSNV site. The genetic variants were annotated bySnpEff v4.1 (Ruden, D. M. et al., Fly 2012, 6:80-92). The NGS data wasdisplayed by generating plots with Circos (v 0.67) (Krzywinski, M. etal., Genome Res. 2009, 19:1639-1645).

Field Sample Collection and Preparation

AIV field samples were collected by inserting Dacron swabs (Fisherbrand,Catalog No. 14-959-97B) into cloaca of poultry objects. The swabs weretransferred into a cryovial containing 5 mL viral transport medium,which was prepared by following WHO guidance. Prior to testing, acryovial containing swab was first shaken by a vortex mixer (IKA MS2 S9Mini Shaker) and then centrifuged under 1,500 g for 30 minutes. Thesupernatant was collected and passed through a membrane filter of 0.2 μmpore size prior to use.

The turkey tissue sample was from a turkey eyelid with gross lesion ofswelling. The tissue sample was minced with sterile scissors in a 20 mLsterile plastic container (Cat No. 14310-684, VWR) containing viraltransport medium at 1:5 (w/v) dilution. The minced tissue wastransferred to a sterile Stomacher bag and homogenized in a Stomacherblender (Model 80, Seward Ltd., UK) for 2-3 minutes. The tissuehomogenate was centrifuged at 1500 rpm for 10 min. The supernatant wasfiltered through a 0.45 μm syringe filter into a polypropylene conicaltube, ready for virus detection.

The results are now described.

Device Operation and Design Based on Tunable Inter-Tubular Distance ofAligned N-MWCNTs

Depending on the type and source of the virus-containing sample, virusparticles need to be released into aqueous suspensions by gentlevortexing, shaking (for swab samples) or tissue homogenization (fortissue samples), before they are introduced into the CNT-STEM (FIG.22A). Large cells or tissue debris were removed by filtering the crudesamples with membrane filters exhibiting pore size of 220 nm or 450 nm(not shown). As the virus suspension flows through the CNT-STEM, virusparticles are efficiently captured by the N-MWCNT arrays whilecontaminants of small size flow through (FIG. 22B). If needed, theviruses (tightly adhered to the carbon nanotubes) can be easilyretrieved/studied after opening the device (FIG. 23A, FIG. 23B). Inorder to synthesize these vertically aligned N-MWCNT on the micro-devicesubstrate, standard semiconductor batch microfabrication techniques wereused to pattern catalyst (iron clusters), followed by selective growthusing aerosol-assisted chemical vapor deposition (AACVD; FIG. 27A andFIG. 24) (Reyes-Reyes, M. et al., Chem. Phys. Lett. 2004, 396:167-173;Villalpando-Paez, F. et al., Chem. Phys. Lett. 2006, 424:345-352).

SEM and TEM images (FIG. 27B through FIG. 27E) as well as Ramanmeasurements with calculated D/G intensity ratios (FIG. 28A and FIG.28B) confirm the presence of N-MWCNTs synthesized directly on thesubstrates (Reyes-Reyes, M. et al., Chem. Phys. Lett. 2004, 396:167-173;Villalpando-Paez, F. et al., Chem. Phys. Lett. 2006, 424:345-352;Sumpter, B. G. et al., ACS nano 2007, 1:369-375). N-MWCNTs were selectedfor their excellent mechanical strength (Yu, M. F. et al., Science 2000,287:637-640) and optimal biocompatibility as reported by a previousstudy (Mihalchik, A. L. et al., Toxicology 2015, 333:25-36). Aftergrowing N-MWCNT arrays on patterned areas of the substrate, the siliconsubstrate was bonded with a PDMS chamber in order to perfectly seal themicro-fluidic chamber without experiencing any leakage.

An important accomplishment of this work is the control of theinter-tubular distance within the CNT arrays so they could matchdifferent virus sizes. In this context, different iron catalystthickness were deposited onto the Si substrates (FIG. 27F through FIG.27J). When the thickness of the iron catalyst thin film increases from 1nm to 12 nm, the density of the iron particles decreases while theparticle diameter increases, thus causing the inter-tubular distance ofN-MWCNTs to increase from 17±6 nm to 325±56 nm. It is also noteworthythat N-MWCNTs consist of concentric tubules exhibiting average diametersof 17-99 nm. In general, the height of the N-MWCNTs also increases overtime, however the growth rate significantly decreases after 20-30minutes of synthesis (FIG. 28C through FIG. 28F).

Performance of size tunable enrichment characterization

In order to validate the size-tunable enrichment capability of theCNT-STEM, fluorescent molecules and fluorescent polystyrene nanospheresof 20 nm, 50 nm, 100 nm, 140 nm, 400 nm, and 1000 nm in diameter weretested and introduced into CNT-STEMs exhibiting different inter-tubulardistances (FIG. 29A and FIG. 30A). FIG. 29B shows the filtrationcharacteristics of CNT-STEMs with 25 nm, 95 nm and 325 nm inter-tubulardistances. They all have a binary separation profile, meaning that for aCNT-STEM with a particular inter-tubular distance, smaller nanoscaleparticles usually penetrate the N-MWCNT structure while larger particlescannot. The particle diameter corresponding to a 50% penetration ratio(the background-corrected fluorescence intensity of the filtrate to thatof the original suspension) was defined as the critical particle size ofthe CNT-STEM with a specific inter-tubular distance (FIG. 31). However,the fluorescence intensity inside the N-MWCNT array is extremely low,maintaining at the same level before and after fluorescein orfluorescence nanospheres flow into the device. This can be explained bythe high optical absorbance of the N-MWCNT forest, reported forvertically aligned carbon nanotube forests as a nearly perfect blackbody absorber (deHeer, W. A. et al., Science 1995, 268:845-847; Mizuno,K. et al., Proc. Natl. Acad. Sci. U.S.A 2009, 106:6044-6047; Yang, Z.-P.et al., Nano Lett. 2008, 8:446-451). Similarly, the viruses inside theN-MWCNT array also elude fluorescence detection (FIG. 32A).

By opening the CNT-STEM device and after observing the N-NWCNT arrayunder SEM, the nanospheres embedded inside the N-MWCNT array could beclearly visualized (FIG. 30B). Thus, in order to separate largenanoscale particles from small contaminates, the inter-tubular distanceof the N-MWCNT can be tuned to be smaller than the target nanoscaleparticles but larger than the contaminants.

Label-Free Capture of Viruses by CNT-STEM

A low pathogenic (LP) avian influenza virus (AIV) (Gao, R. et al., NweEngl. J. Med. 2013, 368:1888-1897; WHO, “World health report—A saferfuture: global public health security in the 21st century,” (Geneve,2007); Yang, Z. Y. et al., Science 2007, 317:825-828) was used as amodel system to characterize and optimize the CNT-STEM performance. Inparticular, the performance of the CNT-STEM was studied using swabsamples of a LPAIV subtype H5N2 (A/chicken/PA/7659/1985) by spikingfreshly propagated viruses into tracheal swabs obtained from specificpathogen-free (SPF) chickens. The size of the H5N2 LPAIV was measured as93±35 nm (fig. S5). When 50 processed swab supernatant containing 10⁷EID₅₀/mL H5N2 LPAIV was introduced into CNT-STEMs of 95 nm inter-tubulardistance, SEM and TEM images clearly showed virus particles welldistributed and efficiently trapped inside the N-MWCNT array FIG. 22Binsets). The CNT-STEM captured viruses are readily detected by on-chipindirect fluorescent antibody (IFA) assay using AIV H5 subtype specificmonoclonal antibody (FIG. 32A, FIG. 32B) (Lu, H. et al., J Veter Sci Med2013, 1:5). In general, CNT-STEMs of smaller inter-tubular distanceshowed stronger fluorescence, thus indicating a higher density of thecaptured virus. However, as explained above, viruses trapped inside theN-MWCNT structures cannot generate fluorescence. Thus the on-chipfluorescence staining can only qualitatively detect the existence of theviruses, but incapable of quantify virus counts within CNT forests.

In order to measure virus capture efficiency, conventional reversetranscription real-time PCR (rRT-PCR) was applied. CNT-STEMs were madewith three different inter-tubular distances of 25 nm, 95 nm and 325 nm.Each CNT-STEM was loaded with 50 μL sample containing 10⁶ EID₅₀/mL H5N2LPAIV. By measuring the original virus titer and that of theflow-through after enrichment with CNT-STEM, virus capture efficiency ofthe CNT-STEMs with 25 nm, 95 nm and 325 nm inter-tubular distances wasmeasured as 96.5±0.5%, 88.0±0.3% and 57.5±0.4%, respectively (FIG. 32C,FIG. 33, FIG. 34).

Virus Concentration and Enrichment

The most commonly used viral surveillance tests are rRT-PCR (Ellis, J.S. et al., Rev. Med. Virol. 2002, 12:375-389) and virus isolation(Eisfeld, A. J. et al., Nat. Protocols 2014, 9:2663-2681; Wood, J. M. etal., Nat. Rev. Micro. 2004, 2:842-847), where a major challenge is toyield true positive results for samples containing virus concentrationsbelow the detection limits. It was investigated whether CNT-STEM couldimprove the virus detection limits of rRT-PCR and virus isolation. Inmany cases, captured viruses need to be retrieved from the device forfurther analysis. In the present case, this has been easily achieved byopening the PDMS chamber of the device, and recovering thevirus-embedded within N-MWCNTs using a pipette tip.

In order to investigate the benefit of CNT-STEM on the overall rRT-PCRassay sensitivity, 1.0 mL H5N2 sample was loaded into CNT-STEMs of 25 nminter-tubular distance. The viruses were enriched, retrieved, andre-suspended in a final volume of 50 μL. The same volume was used forconventional rRT-PCR without virus enrichment. After the CNT-STEMenrichment, rRT-PCR detected AIV in all samples (6/6) with originaltiter as low as 1 EID₅₀/mL, while without using the CNT-STEM, therRT-PCR detection limit was measured as 102 EID₅₀/mL for the same AIVsamples (FIG. 32D, FIG. 33, FIG. 35). Therefore, the CNT-STEM improvesthe overall rRT-PCR detection limit by at least two orders of magnitude.To exclude the potential effect of N-MWCNT in rRT-PCR, the same amountof N-MWCNTs was added inside the CNT-STEM into the rRT-PCR reaction, andit was found that the N-MWCNTs do not exhibit adverse effects (FIG. 36).

Virus isolation remains the “gold standard” for AIV diagnostics(Eisfeld, A. J. et al., Nat. Protocols 2014, 9:2663-2681). For thisprocedure, viable intact virus particles are inoculated into anembryonated chicken egg (ECE) and kept under proper conditions for viruscultivation. This procedure fails when the original virus concentrationis too low or the viruses are non-viable or non-proliferable. Therefore,it was investigated whether CNT-STEM enriched virus samples can bedirectly used for virus isolation to study if the trapped viruses areviable, and then if the enrichment procedure can potentially improve thewell-established virus isolation procedure (FIG. 37A). In this context,the H5N2 AIV was prepared in three serial dilutions in titers of 10⁴EID₅₀/mL, 10³ EID₅₀/mL, and 10² EID₅₀/mL. After 72 hours postinoculation in ECEs, viruses were collected from the allantoic fluid andDot-ELISA assay applied using antibody against AIV H5 antigen to testfor the existence of viruses (FIG. 37B). The successful virus isolationrates were measured as 100%, 100%, and 90% for CNT-STEM processedsamples of original virus titers of 10⁴ EID₅₀/mL, 10³ EID₅₀/mL, and 10²EID₅₀/mL, respectively (FIG. 37C). For those samples without CNT-STEMpreparation, the corresponding virus isolation rates were determined as100%, 50%, and 0%, respectively. Therefore, the CNT-STEM retains thevirus viability and significantly improves the virus isolation rate,while the N-MWCNTs do not interfere with the virus cultivation process.

Unknown Virus Enrichment and Detection by NGS

While NGS does not require prior knowledge of pathogens, the combinationof CNT-STEMs for virus enrichment and NGS for virus identification canbe a unique and powerful approach for discovering unknown/emergingviruses. Normally NGS requires starting genetic materials in microgramrange with high purity in a small volume of tens of microliters(Acevedo, A. et al., Nat. Protocols 2014, 9:1760-1769), which isprohibitive for field samples of low virus count and highlycontaminated. In order to explore the feasibility and develop apractical pipeline of the CNT-STEM for these field conditions, the H5N2LPAIV strain that had been tested in the present study was used toprepare mimic field samples. Although this is an AIV strain isolated in1985, its whole genome has not been sequenced before. Freshly propagatedviruses were spiked into tracheal swabs obtained from SPF chickens to afinal virus titer of 10⁷ EID₅₀/mL titer. Then 250 μL of the preparedsample was loaded into a CNT-STEM of 95 nm inter-tubular distance andRNA extracted into a final volume of 50 μL for NGS analysis. Comparedwith control RNA extracted from 50 μL original H5N2 sample, both theconcentrations of the total RNA and the converted cDNA were higher afterthe CNT-STEM enrichment and concentration (RNA: 870±50 pg/μL versus144±34 pg/μL, cDNA: 3.8 nM versus 0.8 nM). The NGS viral reads increasedfrom 2.9% (37,627 reads) to 90.6% (1,175,537 reads), thus correspondingto an enrichment factor of ˜600, and indicating that the CNT-STEMremoved most of the contamination from the chicken host at the same time(FIG. 38A). For the CNT-STEM processed sample, by following thebioinformatics pipeline in FIG. 26, the viral reads by NGS were de novoassembled into eight single contiguous sequences (contigs) with a˜10⁵×coverage. The nucleotide BLAST search to Genbank (nr/nt database)shows the assembled sequences form the complete genome of theunsequenced H5N2 LPAIV strain (FIG. 38B, FIG. 39). High sequencecoverage allowed for the identification of 38 intrahost variants,including 35 intrahost single nucleotide variation (iSNV) sites, 2intrahost multiple nucleotide variation (iMNV) sites, and 1 deletionsite. By searching through sequenced AIV strains in Genbank, the closeststrain is H5N2 AIV strain A/mallard/Wisconsin/411/1981 isolated frommallard chicken in Wisconsin, USA in 1981. Phylogenetic analysis of HAand NA gene suggested this H5N2 strain (A/chicken/PA/7659/1985) belongsto the same branch of H5N2 strains isolated during 1980s in the Easternand Midwestern United States (FIG. 38C, FIG. 40A, FIG. 40B). Thisunsequenced H5N2 strain was named A/chicken/PA/7659/1985 and depositedinto the NCBI database under KP674444-KP674451 (8 segments, completesequences). This H5N2 strain has the mono-basic cleavage site(PQRETR/GLF) in the HA gene, indicating it is a LPAIV, which can growonly in limited areas of the poultry host (Alexander, D. J. et al.,Avian Influenza. (Blackwell Publishing Ltd., 2009), pp. 217-237).

Field Sample Validation—a Case Study of Avian Influenza Surveillance

In order to validate the new approach for real field samples, a cloacalswab pool collected from five ducks during a 2012 AIV surveillance inPennsylvania were applied. The sample was previously detected as AIVtype A positive by rRT-PCR. Without any virus purification andpropagation, 1.0 mL of the total ˜5 mL suspension of the duck swabsample was enriched and concentrated by a CNT-STEM of 95 nminter-tubular distance. Measured by rRT-PCR, the CNT-STEM increasedvirus titer from 6×10² EID₅₀/mL to 2×10⁴ EID₅₀/mL (FIG. 41). NGS and denovo sequence assembly yielded 8 AIV contigs in complete lengths (FIG.42A), but no AIV related contig was discovered in the sample withoutCNT-STEM enrichment. A nucleotide BLAST search of Genbank (nr/ntdatabase) showed the sequenced AIV was an unsequenced strain and haddifferent homologies to other reported strains, with ˜99% similaritiesto the closest strains (FIG. 45A, FIG. 45B). Phylogenetic analysisindicated the sample is an emerging H11N9 strain. It is closest to twoH11N9 strains A/duck/MN/Sg-00118/2007 (H11N9) andA/pintail/MN/Sg-00149/2007(H11N9) isolated in Minnesota, USA in 2007(FIG. 42B, FIG. 43, FIG. 44A, FIG. 44B). It was named“A/duck/PA/02099/2012 (H11N9)” and the sequence was deposited in theNCBI database under KR870234-KR870241 (8 segments, complete sequences).The H11N9 strain was further confirmed by USDA-NVSL (Ames, Iowa) throughserological tests.

Field Sample Validation—a Case Study of an Unknown Turkey Virus

To verify the utility of the novel method with a truly clinically“unknown” virus, the CNT-STEM was used to process an eyelid tissuehomogenate from a clinical case of a turkey reported to the Penn StateAnimal Diagnostic Laboratory in the summer of 2014. The turkeys had asymptom of blepharoconjunctivitis that had nodules and swollen lesionsand was suspected to be caused by a viral agent. Various common testsfor virus identification based on the symptoms of the infected turkeys,such as general serologic tests (e.g. fluorescent antibody, agar gelimmunodiffustion, hemagglutination-inhibition, virus neutralization),and molecular assays (e.g. PCR) came out negative. CNT-STEM was usedwith NGS as the last resort. First, 5 mL tissue homogenate was filteredthrough a membrane filter of 0.45 μm pore size. Then 750 μL filtrate wasenriched and concentrated to 50 μL by a CNT-STEM of 25 nm inter-tubulardistance and analyzed by NGS. From the CNT-STEM processed sample, 3.81%of the total NGS reads were viral reads (50,076/1,263,289), in contrastto only 0.001% viral reads (17/1,626,134) from 50 μL of the originalmembrane filter tissue filtrate without CNT-STEMenrichment/concentration. The NGS reads represent an enrichment factorof 3.8×10³. After assembly, two viral contigs were obtained with anaverage coverage of 1,056. The nucleotide BLAST search identified thisputative viral agent as a new variant strain of infectious bursaldisease virus (IBDV) with less than 94-95% similarity to other reportedIBDV strains in the USA (FIG. 45A). iSNV analysis identified 4 iSNVsites, where 2 iSNV resulted in amino acid changes. Phylogeneticanalysis and BLAST search results indicated that this is indeed a novelstrain (IBDV/turkey/PA/00924/14), close to an IBDV strain of serotypes 2isolated from turkeys in Ohio, USA (FIG. 45B, FIG. 46, FIG. 47A, FIG.47B). Serotype 2 is relatively rare for sequenced and identified IBDVs,and it is suspected this is the reason why initial serologic andmolecular tests failed to identify the virus. Moreover, the viral agentwas observed under TEM, and it consisted of “birnavirus-like” particlesof ˜65 nm in diameter, well matched with the IBDV identification (FIG.8B inset). The virus was named “MDV/turkey/PA/00924/14”, and thesequence deposited with NCBI database under KP642112 (Segment A) andKP642111 (Segment B).

Carbon nanotubes are among the strongest materials on earth (Yu, M. F.et al., Science 2000, 287:637-640; Qian, D. et al., Appl. Mech. Rev.2002, 55:495-533). The strength and stiffness of N-MWCNTs are comparableto pristine MWCNTs (Ganesan, Y. et al., ACS Nano 2010, 4:7637-7643).Since filtration is mainly a mechanical process, the high stiffness ofthe constructing nanomaterial enables the fabrication of a device withextremely high porosity up to 95% while still maintaining structureintegrity during filtration. Therefore, the robustness of CNTs and theextremely high porosity of the N-MWCNT arrays distinguish the presentCNT-STEM technology from other existing ultrafiltration techniques; atleast two orders of magnitude lower in normalized flow resistancecompared with commercial ultrafiltration membranes (FIG. 48). This highporosity is critical for reducing flow resistance, preventing deviceclogging, and decreasing CNT material usage to minimize negative effectin downstream virus analyses, all of which empower the CNT-STEM as apoint-of-care platform for efficient virus sample preparation fromanimal and human samples.

It is also noteworthy that the overall success rate from devicefabrication to testing is 76.8% out of 228 fabricated devices for thesestudies. In FIG. 49, the device yield and reliability were recorded andanalyzed during the CNT-STEM fabrication, assembly and testing. Theoverall success rate from device fabrication to testing is 76.8% out of228 fabricated devices. During device fabrication, the PDMS top chamberwas aligned to the N-MWCNT forest patterns by naked eyes before bonding,16 out of 228 devices (7.0%) failed because of the misalignment.Although the N-MWCNT forest structure can withstand pressure and forcesduring normal device operation, when it hits a hard surface duringfabrication, it can still “crash”. This kind of mishandling accounts for2.6% of overall device failure. Finally prior to virus filtration, theflow rate was measured during the PBS wash. 31 out of 206 devices werefound to have a leakage problem, which presented 58.5% (31/53) of allfailure devices. The leakage is believed to be caused by somemicro-scale damages of N-MWCNT structures, which compromised theintegrity of the N-MWCNT porous wall and too miniscule to be observedunder an optical microscope directly. To improve the yield of thedevice, some custom-made jigs or tools can be designed for automatichandling during the device fabrication, assembly and testing.

Device failing is due to leakage (13.6%), misalignment of PDMS-CNTs(7.0%) and N-MWCNT structure inhomogeities (2.6%). However, all thesecan be improved by further microfabrication tuning. For leakage, asimple and effective method was developed to evaluate it (beforeintroducing real samples), by measuring the flow rate of buffer solutionthrough the CNT-STEM device.

The tunable range of the inter-tubular distance of N-MWCNT (17-325 nm)spans the majority of the virus size spectrum, and provides uniqueflexibility in device design/fabrication able to reach the bestperformance for different viruses. In order to prepare samples for NGS,it is preferable to use CNT-STEM with larger inter-tubular distance ifhost ribosome RNA (rRNA) is a concern; larger inter-tubular distancewill not trap ribosomes (˜20 nm in diameter). Thus, CNT-STEM with 95 nminter-tubular distance was used for the AIV samples targeted for NGSanalysis. This is also justified for mimicking H5N2 swab samples: therRNA reads reduced from 985,397 (41.7% of total reads) to 33,735 (2.6%of total reads) after the CNT-STEM sample preparation. For viruses ofsmall size or samples with unknown viruses, it is more preferable totest viruses with devices of smaller inter-tubular distance. CNT-STEM of25 nm inter-tubular distance was used to enrich and concentrate unknownviruses from the turkey eyelid tissue sample, and it turned out that theisolated IBDV was smaller than the previously tested AIV (65 nm vs 93nm).

It has been reported that high concentration of CNTs can inhibit PCRwhile low concentration of CNTs may enhance it. The present experimentsdemonstrate there was no noticeable effect of N-MWCNT on the Ct valuesof the rRT-PCR. The weight of N-MWCNTs inside each CNT-STEM was measuredas 26 μg, which corresponds to a final concentration of 0.5 μg/μL in therRT-PCR reaction mixture. The concentration is consistent with thepreviously reported CNT concentration ranges that have no effect or canenhance PCR.

In both the rRT-PCR virus detection and the ECE virus isolationexperiments, the improvement correlates with the volume ratio of theoriginal sample to that of the re-suspended sample after enrichment,which underlines the importance of the optimal sample concentrationprovided by the CNT-STEM. Concentration effects can account for a largepart of the improvement of rRT-PCR and virus isolation, since thecontaminating materials do not significantly affect the highly specificrRT-PCR virus detection and they are non-proliferable in embryonatedchicken eggs. However, the contaminant removal and sample concentrationare key for the whole genome sequencing using NGS, because randomprimers are used that do not distinguish viral targets from othercontaminating genetic materials.

The CNT-STEM reported here provides a unique platform of ananomaterial-integrated microfluidic device for label-free enrichmentand concentration of viruses from field samples. By engineering thebottom-up synthesis process, N-MWCNTs arrays were selectively grown ondevice substrates and then integrated directly into microfluidicdevices. This combined bottom-up synthesis and top-down microfabricationmakes the production of the device potentially scalable and low cost.The unique properties of the vertically aligned N-MWCNT enable theCNT-STEM to enrich viable virus particles, and remove host andenvironmental contaminants in a highly efficient way. The tunable sizerange of the CNT-STEM covers the size of most of the reported viruses.This novel technology was demonstrated to significantly improve currentrRT-PCR and virus isolation in avian influenza virus surveillance. Moreimportantly, it enables genomic sequencing using NGS directly from realfield samples without virus amplification. Since neither CNT-STEM basedvirus sample preparation nor NGS requires prior knowledge of the virusesinside the sample, this combination provides a unique and powerfulapproach for novel and emerging virus discovery, thus significantlycontributing to the control and eradication of viral infectiousdiseases.

Example 4: Plant Plum Pox Virus (PPV) Enrichment

A feasibility study of PPV enrichment was performed by CNT-STEM.CNT-STEM with 25 nm inter-tubular distance was used to enrich PPVsamples. The enrichment results were measured by RT-PCR. FIG. 50A showsthe Ct values of PPV serial dilution samples after being processed bycurrent a USDA protocol and by CNT-STEM. Overall, CNT-STEM has betterPPV enrichment performance compared to the current USDA protocol. UnderPPV with 100× dilution, CNT-STEM improved the Ct value by ˜7, whichcorresponds to ˜100 times better yield compared to the current USDAstandard protocol. This preliminary study demonstrated the feasibilityof CNT-STEM as an effective sample preparation platform for PPV fieldsample preparation.

The materials and methods of the feasibility study are now described.

Leaf Sample Preparation

A set of plant grinding bags were labeled for the number of samples tobe tested, wherein each laboratory sample should consist of no more than8 leaves. The leaves were stacked on top of each other and a portion ofthe leaves were torn nearest to the petiole end, along the mid rib onthe remaining side of the leaves. 0.3 g of each sample were weighed andplaced in the corresponding grinding bag between the mesh layers of thegrinding bag. Samples were kept on ice.

Pre-chilled GEB4 grinding buffer was added to each bag at 1:10 ratio(tissue weight in g:buffer volume in mL). A tissue homogenizer devicewas used to grind the leaves into sap from the outside of the bag. Aftergrinding, the bags of ground sap were kept in ice until loading. Atleast 500 μL of ground sap into a 1.6 mL disposable microcentrifugetube. All processed samples should be loaded into a prepared platewithin 1-2 hours of grinding.

One-Step RT-PCR

All reagents, primers, and probes were thawed at room temperature,except the SuperScript® III RT/Platinum® Taq Mix and RNaseOUT were kepton ice. All thawed reagents were vortexed at a setting of 7-10 untilwell homogenized. Vortexed tubes were spun for 15-30 seconds at >10,000rpm in a bench-top microcentrifuge and kept on ice.

RT-PCR Set-Up

RT-PCR TaqMan was performed with Invitrogen's SuperScript® III Platinum®One-Step qRT-PCR Kit. The kit consists of 2× Reaction Mix (containingRT-PCR buffer, 3 mM Mg and dNTPs), 50 mM MgSO₄ and SuperScript® IIIRT/Platinum® Taq Mix. The kit also contains ROX Reference Dye, which wasnot used. Master Mix preparation and aliquoting must be done in adecontaminated PCR workstation/enclosure on a new disposable lab mat.

A Cepheid cooling block was removed from a 4° C. refrigerator and placedin the PCR workstation. 20 μL of PPV Master Mix was prepared for eachsample plus 4-6 control tubes plus 1 extra mix for every 10 reactionsneeded. For example, for 20 reactions, 22 volumes of PPV Master Mix wasprepared. Each volume of PPV Master Mix contained: 3.5 μL MolecularGrade Water; 12.54 μL 2× Reaction Mix; 1.5 μL 50 mM MgSO₄; 1 μL 5 μM SchFRD-REV primer mix; 0.5 μL 5 μM Sch probe; 0.5 μL 40u/μL RNaseOUT; and0.5 μL SuperScript® III/Platinum® Taq Mix. Master Mix was mixed bypipetting gently up and down 2-3 times and kept on ice. SmartCycler®tubes (25 μL) were placed into the Cepheid cooling block, and the 20 μLvolumes of Master Mix were pipetted into each tube. The tubes were takento the PCR thermocycler station and 5 μL of RNA sample or control wereadded to the appropriate tubes to yield a total reaction volume of 25μL.

The disclosures of each and every patent, patent application, andpublication cited herein are hereby incorporated herein by reference intheir entirety. While this invention has been disclosed with referenceto specific embodiments, it is apparent that other embodiments andvariations of this invention may be devised by others skilled in the artwithout departing from the true spirit and scope of the invention. Theappended claims are intended to be construed to include all suchembodiments and equivalent variations.

The invention claimed is:
 1. An enrichment platform device forsize-based, label-free capture of particles in sample solution, thedevice comprising: a substrate; vertically-aligned carbon nanotubesarrays (VACNT); and a cover having at least one inlet and at least oneoutlet; wherein the VACNT is attached to the substrate, the cover bondsto the substrate to seal the VACNT within the cover, and sample solutionenters via the at least one inlet, passes over the VACNT, and exits viathe at least one outlet, whereupon particles in the sample solution arecaptured by the gaps between the VACNT based on size: and wherein theVACNT is attached to a single layer of metal catalyst thin film that isdirectly attached to the substrate, each VACNT being separated by aVACNT gap having a VACNT gap size, wherein at least one VACNT gap sizeis different from another VACNT gap size, wherein each VACNT gap size issized to capture a particle having a particle size commensurate with theVACNT gap size so that different sized particles are releasably capturedin an appropriate sized VACNT gap, the capture mechanism consistingessentially of the particles fitting in the VACNT gaps via mechanicalinterference.
 2. The enrichment platform device of claim 1, wherein thesubstrate comprises material selected from the group consisting of:silicon, glass, sapphire, metals, and polymers.
 3. The enrichmentplatform device of claim 1, wherein the cover comprises materialselected from the group consisting of: plastics, metals, glass,sapphire, polymers, and polydimethylsiloxane (PDMS).
 4. The enrichmentplatform device of claim 1, wherein the cover is removable.
 5. Theenrichment platform device of claim 1, wherein the VACNT comprisesingle-walled CNT, double-walled CNT, multi-walled CNT, and/orcombinations thereof.
 6. The enrichment platform device of claim 1,wherein the VACNT is nitrogen-doped VACNT, boron-doped VACNT,silicon-doped VACNT, aluminum doped VACNT, phosphorus-doped VACNT,lithium-doped VACNT, and/or combinations thereof.
 7. The enrichmentplatform device of claim 1, wherein the VACNT gap size is between 1 nmand 500 nm.
 8. The enrichment platform device of claim 1, wherein thedevice is a microfluidic device.
 9. The enrichment platform device ofclaim 1, wherein the device is a handheld device.
 10. The enrichmentplatform device of claim 1, wherein the metal catalyst thin film is aniron-catalyst thin film, a nickel-catalyst thin film, or acobalt-catalyst thin film.
 11. The enrichment platform device of claim1, wherein the metal catalyst thin film has a thickness that isadjustable to tune gap size, diameter, and density of the VACNT.
 12. Theenrichment platform device of claim 1, wherein the VACNT comprisescarbon nanotube (CNT) precursor material deposited on the metal catalystthin film.
 13. The enrichment platform device of claim 1, wherein theprecursor material comprises benzylamine.
 14. The enrichment platformdevice of claim 1, wherein the metal catalyst thin film has a thicknessthat is less than 20 nm.